JP5137400B2 - Peptides for diagnosing and treating amyloid-related diseases, antibodies thereto, and methods of use thereof - Google Patents

Description

  The present invention relates to peptides and antibodies thereto that can be used to diagnose, prevent, and treat amyloid-related diseases such as type II diabetes and Alzheimer's disease.

  Amyloid deposition (also called amyloid plaque formation) is associated with a variety of unrelated pathological conditions, including Alzheimer's disease, prion-related brain damage, type II diabetes, familial amyloidosis and light chain amyloidosis. It is a central feature.

  The amyloid material is composed of a dense network of stiff unbranched proteinaceous fibrils of indefinite length with a diameter of about 80-100 cm. Amyloid fibrils contain a core structure of polypeptide chains arranged in an antiparallel or parallel β-pleated sheet whose major axis is orthogonal to the major axis of the fibrils [Both et al. (1997) Nature 385: 787-93; Glenner (1980), N.M. Eng. J. et al. Med. 302: 1283-92; Balbach et al. (2002), Biophys J. et al. 83: 1205-16].

  About 20 amyloid fibril proteins have so far been identified in vivo and correlated with specific diseases. Although these proteins have little amino acid sequence homology to each other, the core structure of amyloid fibrils is essentially the same. This common core structure of amyloid fibrils, and the presence of common substances in amyloid deposits, data that characterize a particular form of amyloid substance may also be related to other forms of amyloid substance, thus It suggests that it can be embodied in template designs to develop drugs for amyloid-related diseases such as type II diabetes, Alzheimer's dementia or Alzheimer's disease, and prion-related brain disorders.

  Furthermore, amyloid deposits do not appear to be inactive in vivo, but rather are in a dynamic state of turnover and may even regress if fibril formation is stopped [Gillmore et al. (1997), Br. J. et al. Haematol. 99: 245-56].

  Accordingly, therapies designed to inhibit amyloid polypeptide production or to inhibit amyloidosis may be useful for treating amyloid-related diseases.

  Inhibition of amyloid polypeptide production-Direct inhibition of amyloid polypeptide production can be achieved, for example, by the use of antisense oligonucleotides, such as antisense oligonucleotides to messenger RNA (mRNA) of human islet amyloid polypeptide. . In vitro, addition of antisense oligonucleotides to islet amyloid polypeptide mRNA or expression of antisense-complementary DNA increases cellular insulin mRNA and protein content, thereby increasing the potential of this method. Effectiveness has been demonstrated [Kulkarni et al. (1996), J. MoI. Endocrinol. 151: 341-8; Novals et al. (1998) Pancreas, 17: 182-6]. However, no experimental results have been clarified to demonstrate the in vivo efficacy of such antisense molecules.

  Inhibition of amyloid fibril formation—Amyloids, including islet amyloid, contain potential stabilizing or protective substances such as serum amyloid P component, apolipoprotein E and perlecan. Treatment with antibodies specific for specific portions of amyloidogenic proteins by blocking their binding to developing amyloid fibrils [Solomon et al. (1997) Proc. Natl. Acad. Sci. USA, 94: 4109-12] can inhibit amyloid formation [Kahn et al. (1999), Diabetes, 48: 241-53].

  The following summarizes current attempts to manipulate drugs that have the ability to destabilize amyloid structures.

  The destabilizing compound-heparin sulfate has been identified as a component of all amyloids, and heparin sulfate is also implicated in the very early stages of amyloid induction associated with inflammation. Kisilevsky and co-workers (Mature Med., 1: 143-148, 1995) have reported that low molecular weights that prevent heparin sulfate from interacting with inflammation-related amyloid precursors and β-peptides of Alzheimer's disease (AD). The use of anionic sulfonate or sulfate compounds has been described. Heparin sulfate has a specific effect on the soluble amyloid precursor (SAA2) to accept the increased β-sheet structure characteristic of the amyloid protein folding pattern. These anionic sulfonate compounds or sulfate compounds have been shown to inhibit heparin-accelerated Aβ fibril formation as monitored by electron micrographs, and pre-formed fibrils in vitro. Could be decomposed. Furthermore, these compounds substantially halted inflammation-related amyloid progression in the spleen of mice in vivo in acute and chronic models. However, the most potent compound [ie poly (vinyl sulfonate)] showed acute toxicity. Similar toxicity has been observed for another compound IDOX (anthracycline 4′-iodo-4′-deoxyxorubicin) that has been observed to induce amyloid reabsorption in immunoglobulin light chain amyloidosis (AL) patients. [Merlini et al. (1995), Proc. Natl. Acad. Sci. USA 92: 2959-63].

  Destabilized antibody-anti-β-amyloid monoclonal antibody has been shown to be effective in disaggregating β-amyloid plaques and preventing the formation of β-amyloid plaques in vitro (US Pat. No. 5,688,561). issue). However, no experimental results have been clarified to demonstrate the effectiveness of such antibodies in vivo.

  The discovery that the addition of a synthetic peptide that breaks the destabilized peptide-β-pleated sheet (“β-sheet breaker”) dissociated the fibrils and prevented amyloidosis [Soto et al. (1998) Nat. Med. 4: 822-6] is particularly promising from a clinical point of view. Briefly, a 5-residue peptide inhibits fibril formation of amyloid β-protein, degrades preformed fibrils in vitro, and is induced by fibrils in cell culture. Death was hindered. Furthermore, this β-sheet disruptor peptide significantly reduced amyloid β-protein deposition in vivo and completely blocked amyloid fibril formation in a rat brain model of amyloidosis.

  The possibility of using small molecules that bind small molecule-amyloid polypeptides and thereby stabilize the original folding of the protein has been attempted in the case of transthyretin (TTR) proteins [Peterson (1998), Proc. Natl. Acad. Sci. USA, 95: 12965-12960; Oza (1999), Bioorg. Med. Chem. Lett. 9: 1-6]. To date, it has been shown that molecules such as thyroxine and flufenamic acid can prevent conformational changes leading to amyloid formation. However, the use of these compounds in animal models has not yet been demonstrated and can be compromised due to the presence in the blood of proteins other than TTR that can bind to these ligands.

  Antioxidants—Another proposed treatment was to take antioxidants to avoid oxidative stress and maintain the amyloid protein in its reduced state (ie, monomers and dimers). The use of sulfite has been shown to yield more stable monomers of TTR both in vitro and in vivo [Altland (1999) Neurogenetics 2: 183-188]. However, a complete characterization of the antioxidant effect has not yet been obtained, and interpretation of the results regarding possible treatments remains difficult.

  As the invention was put into practice, we found that contrary to the teaching of US Pat. No. 6,359,112 (Kapuriotu), peptide aggregation on amyloid fibrils is dominated by aromatic interactions. ing. Such knowledge allows efficient and accurate design of peptides that can be used to diagnose and treat amyloid-related diseases.

  According to one aspect of the present invention, a peptide comprising the amino acid sequence XY or YX, wherein X is an aromatic amino acid, Y is an amino acid other than glycine, and at least 2 and 15 or less Peptides having the amino acid length of

  According to another aspect of the present invention, a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12 to 19, 27 to 45, 112 to 123, 125, and 127, wherein at least two Peptides having a length of no more than amino acids are provided.

  According to still another aspect of the present invention, there is provided a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 12 to 19, 27 to 45, 112 to 123, 125 and 127, wherein at least two Peptides having a length of 15 amino acids or less are provided.

  According to yet another aspect of the present invention, there is provided a peptide selected from the group consisting of SEQ ID NOs: 4, 12-19, 27-45, 112-123, 125 and 127.

  According to a further aspect of the present invention, there is provided a method for treating or preventing an amyloid-related disease in an individual, comprising the amino acid sequence XY or YX, wherein X is an aromatic amino acid and Y is an amino acid other than glycine. A method comprising: providing an individual with a therapeutically effective amount of at least two peptides having a length of no more than 15 amino acids.

  According to further features in preferred embodiments of the invention described below, the peptide is an active ingredient of a pharmaceutical composition that also includes a physiologically acceptable carrier.

  According to still further features in the described preferred embodiments the peptide is expressed from the nucleic acid construct.

  According to yet a further aspect of the invention, a peptide comprising the amino acid sequence XY or YX, wherein X is an aromatic amino acid and Y is an amino acid other than glycine, comprising at least two There is provided a pharmaceutical composition for treating or preventing amyloid-related diseases comprising a peptide having a length of 15 amino acids or less as an active ingredient and comprising a pharmaceutically acceptable carrier or diluent.

  According to still further features in the described preferred embodiments at least one amino acid of the peptide having a length of at least 2 and not more than 15 amino acids is a D stereoisomer.

  According to still further features in the described preferred embodiments at least one amino acid of the peptide having a length of at least 2 and not more than 15 amino acids is an L stereoisomer.

  According to still further features in the described preferred embodiments the peptide is 2 amino acids in length and Y is a β-sheet breaker amino acid.

  According to still further features in the described preferred embodiments the peptide is as set forth in SEQ ID NO: 145.

  According to still further features in the described preferred embodiments the peptide is 3 amino acids in length, Y is an aromatic amino acid and the amino acid residue attached to the amino acid sequence XY or YX Is a β-sheet breaker amino acid.

  According to still further features in the described preferred embodiments the β-sheet breaker amino acid is at the C-terminus of the peptide.

  According to still further features in the described preferred embodiments the peptide has a length of at least 3 amino acids and comprises a thiolated amino acid at its N-terminus.

  According to yet a further aspect of the invention, a peptide comprising the amino acid sequence XY or YX, wherein X is an aromatic amino acid and Y is an amino acid other than glycine, comprising at least two Nucleic acid constructs comprising polynucleotide segments encoding peptides having a length of 15 amino acids or less are provided.

  According to still further features in the described preferred embodiments the nucleic acid construct further comprises a promoter.

  According to a further aspect of the present invention, there is a peptide comprising the amino acid sequence XY or YX (where X is an aromatic amino acid and Y is an amino acid other than glycine), at least two of which are 15 An antibody or antibody fragment comprising an antigen recognition region capable of binding to a peptide having a length of no more than amino acids is provided.

  According to still further features in the described preferred embodiments Y is an amino acid having no polar charge selected from the group consisting of serine, threonine, asparagine, glutamine and their natural derivatives.

  According to still further features in the described preferred embodiments Y is a β-sheet breaker amino acid.

  According to still further features in the described preferred embodiments the β-sheet breaker amino acid is a naturally occurring amino acid.

  According to still further features in the described preferred embodiments the naturally occurring amino acid is selected from the group consisting of proline, aspartic acid, glutamic acid, glycine, lysine and serine.

  According to still further features in the described preferred embodiments the β-sheet breaker amino acid is a synthetic amino acid.

  According to still further features in the described preferred embodiments the synthetic amino acid is a Cα-methylated amino acid.

  According to still further features in the described preferred embodiments the Cα-methylated amino acid is α-aminoisobutyric acid.

  According to still further features in the described preferred embodiments the peptide is a linear or cyclic peptide.

  According to still further features in the described preferred embodiments the peptide is selected from the group consisting of SEQ ID NOs: 4, 12-19, 27-45, 112-123, 125 and 127.

  According to still further features in the described preferred embodiments the peptide has a length of at least 4 amino acids and comprises at least 2 serine residues at its C-terminus.

  According to still further features in the described preferred embodiments the peptide has a length of at least 3 amino acids and at least one amino acid of the peptides other than XY is serine, threonine, asparagine, glutamine and A non-polar amino acid selected from the group consisting of their natural derivatives.

  According to still further features in the described preferred embodiments the peptide has a length of at least 3 amino acids and at least one amino acid of the peptides other than XY is a β-sheet breaker amino acid .

  According to still further features in the described preferred embodiments the β-sheet breaker amino acid is a naturally occurring amino acid.

  According to still further features in the described preferred embodiments the naturally occurring amino acid is selected from the group consisting of proline, aspartic acid, glutamic acid, glycine, lysine and serine.

  According to still further features in the described preferred embodiments the β-sheet breaker amino acid is a synthetic amino acid.

  According to still further features in the described preferred embodiments the synthetic amino acid is a Cα-methylated amino acid.

  According to still further features in the described preferred embodiments the Cα-methylated amino acid is α-aminoisobutyric acid.

  According to still further features in the described preferred embodiments the β-sheet breaker amino acid is located downstream of XY in the peptide.

  According to still further features in the described preferred embodiments the β-sheet breaker amino acid is located upstream of XY in the peptide.

  According to still further features in the described preferred embodiments the peptide has a length of at least 3 amino acids, at least one amino acid of the peptide is a positively charged amino acid, and at least 1 of the peptide An amino acid residue is an amino acid having a negative charge.

  According to still further features in the described preferred embodiments the positively charged amino acid is selected from the group consisting of lysine, arginine, and natural and synthetic derivatives thereof.

  According to still further features in the described preferred embodiments the negatively charged amino acid is selected from the group consisting of aspartic acid, glutamic acid, and natural and synthetic derivatives thereof.

  According to yet a further aspect of the invention, a peptide comprising the amino acid sequence XY or YX, wherein X is an aromatic amino acid and Y is an amino acid other than glycine, comprising at least two Treating or preventing an amyloid-related disease comprising an antibody or antibody fragment having an antigen recognition region capable of binding to a peptide having a length of 15 amino acids or less as an active ingredient, and comprising a pharmaceutically acceptable carrier or diluent A pharmaceutical composition is provided.

  According to yet a further aspect of the present invention, there is provided a method for treating or preventing an amyloid-related disease in an individual comprising the amino acid sequence XY or YX (where X is an aromatic amino acid and Y is other than glycine). A therapeutically effective amount of an antibody or antibody fragment having an antigen recognition region capable of binding to a peptide having a length of at least 2 and not more than 15 amino acids. Is provided.

According to yet a further aspect of the invention, there are provided peptides having the general formula:
Where C ^* is a chiral carbon having the D configuration.
R ₁ and R ₂ are each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carboxy, C-thiocarb;
R ₃ is selected from the group consisting of hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, halo and amine; and R ₄ is alkyl.

According to yet a further aspect of the invention, there is provided a method of treating or preventing an amyloid-related disease in an individual, comprising providing the individual with a therapeutically effective amount of a peptide having the general formula:
Where C ^* is a chiral carbon having the D configuration.
R ₁ and R ₂ are each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carboxy, C-thiocarb;
R ₃ is selected from the group consisting of hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, halo and amine; and R ₄ is alkyl.

According to still further features in the described preferred embodiments R ₄ is methyl.

According to still further features in the described preferred embodiments R ₁ and R ₂ are each hydrogen and R ₃ is hydroxy.

  According to still further features in the described preferred embodiments the peptide is a cyclic peptide.

  The present invention addresses various drawbacks of currently known forms by providing novel peptides, compositions and methods that can be used to diagnose and treat amyloid-related diseases such as type II diabetes. Has been successful in dealing with it.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples provided herein are for purposes of illustration only and are not intended to limit the invention.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is described herein by way of example with reference to the accompanying drawings. The details shown in the drawings are by way of example only and are provided to illustrate the principles and conceptual features of the invention in a manner deemed most effective and understandable, only for the purpose of illustrating preferred embodiments of the invention. Is. In this regard, the structural details of the present invention have been described in detail only to the extent necessary for a basic understanding of the present invention, but upon examination of the present specification and drawings together, those skilled in the art will be able to It will be clear how to actually embody some aspects of the invention.
FIG. 1 is a schematic diagram showing the self-association ability and hydrophobicity of peptides derived from a large number of amyloid proteins, as estimated using the Kyte and Dolittle scales. Note that there is no correlation between the hydrophobicity of the analyzed peptides and the ability to form amyloid. The only obvious measure for potential amyloid fibril forming ability in this group of peptides is the combination of aromaticity and minimum length.
Figures 2a-c schematically show amyloid binding with the inhibitory aromatic reagents Ro47-1816 / 001 (Figure 2a), Thioflavin T (Figure 2b) and CR dye (Figure 2c).
Figures 3a-c are schematics of primary sequence comparisons between human IAPP and rodent IAPP and the synthetic peptides of the present invention. FIG. 3a is a sequence alignment of human IAPP and rodent IAPP. The block part shows a partial sequence of 7 amino acids showing a large discrepancy between the sequences. The “basic amyloid-forming unit” is shown in bold and underlined. FIG. 3b shows the chemical structure of the wild type IAPP peptide (SEQ ID NO: 1). FIG. 3c shows the primary sequence and SEQ ID NO of the peptide derived from the basic amyloid-forming unit.
Figures 4a-b are graphs showing the absorbance of light at 405 nm as a function of time during fibril formation, and thus the absorbance reflecting the aggregation kinetics of IAPP-derived peptides. The following symbols are used: black square-N1A, white circle-G3A, black circle-wild type, white triangle-L6A, white square-I5A, and black triangle-F2A.
FIG. 5 is a histogram showing the average particle size of associated IAPP peptides and derivatives as measured by light scattering. Each column represents the result of 3 to 5 independent measurements.
Figures 6a-n are photomicrographs showing the binding of Congo red to pre-associated IAPP peptides. Micrographs in normal and polarized fields are shown for each of the following laid peptide suspensions, respectively: N1A peptide (FIGS. 6a-6b), F2A peptide (FIGS. 6c-6d), G3A peptide (FIG. 6e-6f), wild-type peptide (FIGS. 6g-6h), I5A peptide (FIGS. 6i-6j), and L6A peptide (FIGS. 6k-6l).
FIG. 7 are electron micrographs of “laying” IAPP peptides and derivatives: N1A peptide (FIG. 7a), F2A peptide (FIG. 7b), G3A peptide (FIG. 7c), wild type peptide (FIG. 7d), I5A peptide ( Fig. 7e), and L6A peptide (Fig. 7f). The scale bar shown represents 100 nm.
FIG. 8a is a nucleic acid sequence alignment of wild type hIAPP and corresponding sequences modified according to bacterial codon usage. The modified base is underlined.
FIG. 8b is a schematic diagram of the pMALc2x-NN vector used for cytoplasmic expression of the 48 kDa MBP-IAPP protein. A V8 Ek cleavage site and a (His) ₆ tag are fused to the C-terminus of the malE tag vector sequence. The factor Xa cleavage site for removing the MBP tag is shown.
FIG. 9 is a protein gel GelCode Blue staining showing bacterial expression and purification of MBP and MBP-IAPP fusion proteins. A bacterial cell extract was prepared and the protein was purified on an amylose resin column. A sample containing 25 μg of protein was added to each of lanes 1-3, whereas 5 μg of protein was added to each of lanes 4-5. Proteins were separated by 12% SDS-PAGE and visualized by GelCode Blue staining. Molecular weight markers are shown on the left. Lane 1-MBP soluble extract induced with 0.5 mM IPGT, Lane 2-MBP-IAPP soluble extract induced with 0.1 mM IPGT, Lane 3-MBP-induced with 0.5 mM IPGT IAPP soluble extract, lane 4-purified MBP, lane 5-purified MBP-IAPP. The arrow indicates MBP-IAPP.
FIG. 10a-b is a dot blot image (FIG. 10a) showing the putative amyloidogenic sequence in hIAPP and its densitometric quantification (FIG. 10b).
FIG. 11 is a graph showing the absorbance of light at 405 nm as a function of time during fibril formation, and thus the absorbance reflecting the aggregation kinetics of the IAPP-derived peptide (SEQ ID NOs: 14-19).
Figures 12a-f are photomicrographs showing congo red binding to pre-associated IAPP peptides. Photomicrographs in polarized fields are shown for each of the following one day aged peptide suspensions: NFLVHSSNN peptide (FIG. 12a), NFLVHSS (FIG. 12b), FLVHSS (FIG. 12c), NFLVH (FIG. 12d), FLVHS. (FIG. 12e) and FLVH (FIG. 12f).
FIGS. 13a-f are electron micrographs of “laying” IAPP peptides. NFLVHSSNN peptides (FIG. 13a), NFLVHSS (FIG. 13b), FLVHSS (FIG. 13c), NFLVH (FIG. 13d), FLVHS (FIG. 13e), and FLVH (FIG. 13f). The scale bar shown represents 100 nm.
14a-f are graphs showing the secondary structure in insoluble IAPP aggregates as measured by Fourier transform infrared spectroscopy. NFLVHSSNN peptides (FIG. 14a), NFLVHSS (FIG. 14b), FLVHSS (FIG. 14c), NFLVH (FIG. 14d), FLVHS (FIG. 14e), and FLVH (FIG. 14f).
FIG. 15 is the chemical structure of a previously reported amyloidogenic peptide fragment of medin [Haggqvist (1999), Proc. Natl. Acad. Sci. USA, 96: 8669-8684].
FIGS. 16a-b are graphs showing the absorbance of light at 405 nm as a function of time during fibril formation, and thus the absorbance reflecting the aggregation kinetics of medin-derived peptides. FIG. 16a shows a short-term kinetic assay. FIG. 16b shows a long-term kinetic assay.
FIGS. 17 a-f are electron micrographs of “laying” medin-derived peptides. NFGSVQFA—FIG. 17a, NFGSVQ—FIG. 17b, NFGSV—FIG. 17c, FGSVQ—FIG. 17d, GSVQ—FIG. 17e, and FGSV—FIG. 17f. The scale bar shown represents 100 nm.
FIGS. 18a-f are photomicrographs showing congo red binding to pre-associated medin-derived peptides. Polarized field micrographs are shown for each of the following laid peptide suspensions: NFGSVQFA—FIG. 18a, NFGSVQ—FIG. 18b, NFGSV—FIG. 18c, FGSVQ—FIG. 18d, GSVQ—FIG. 18e, and FGSV— FIG. 18f.
Figures 19a-c show the effect of alanine mutations on the amyloidogenic properties of the amyloidogenic fragment of the hexapeptide of medin. FIG. 19a is a graph showing the absorbance of light at 405 nm as a function of time during fibril formation, and hence the absorbance reflecting the aggregation kinetics of the medin derived alanine variant. FIG. 19b is an electron micrograph of an alanine variant derived from a “laying” medin and the scale bar represents 100 nm. FIG. 19c is a photomicrograph showing the binding of Congo red to pre-associated mesin-derived peptide variants.
Figures 20a-b are the amino acid sequence of human calcitonin (Figure 20a) and the chemical structure of an amyloidogenic peptide fragment of human calcitonin (Figure 20b). Residues 17 and 18 that are important for the calcitonin oligomerization state and hormonal activity are underlined [Kazantzis (2001), Eur. J. et al. Biochem. 269: 780-791].
FIGS. 21 a-d are electron micrographs of “laying” calcitonin derived peptides. DFNKF—FIG. 21a, DFNK—FIG. 21b, FNKF—FIG. 21c, and DFN—FIG. 21d. The scale bar shown represents 100 nm.
Figures 22a-d are photomicrographs showing the binding of Congo red to pre-associated calcitonin derived peptides. Photomicrographs in the polarized field are shown for each of the following laid peptide suspensions: DFNKF—FIG. 22a, DFNK—FIG. 22b, FNKF—FIG. 22c, and DFN—FIG. 22d.
FIG. 23 is a graph showing the secondary structure of insoluble calcitonin aggregates as measured by Fourier transform infrared spectroscopy.
Figures 24a-c show the effect of alanine mutations on the amyloidogenic properties of the amyloidogenic fragment of the calcitonin pentapeptide. FIG. 24a is an electron micrograph of an alanine variant derived from “laying” calcitonin. The scale bar represents 100 nm. FIG. 24b is a photomicrograph showing the binding of Congo red to pre-associated calcitonin derived peptide variants. FIG. 24c is a graph showing the secondary structure of the mutant peptide as measured by Fourier transform infrared spectroscopy.
FIG. 25 is an electron micrograph showing self-association of “laying” lactotransferrin-derived peptide. The scale bar represents 100 nm.
FIG. 26 is an electron micrograph showing self-association of a “laying” serum amyloid A protein-derived peptide. The scale bar represents 100 nm.
FIG. 27 is an electron micrograph showing self-association of “laying” BriL-derived peptides. The scale bar represents 100 nm.
FIG. 28 is an electron micrograph showing self-association of “laying” gelsolin derived peptides. The scale bar represents 100 nm.
FIG. 29 is an electron micrograph showing self-association of “laying” serum amyloid P-derived peptide. The scale bar represents 100 nm.
FIG. 30 is an electron micrograph showing self-association of “laying” immunoglobulin light chain derived peptides. The scale bar represents 100 nm.
FIG. 31 is an electron micrograph showing self-association of “laying” cystatin C-derived peptide. The scale bar represents 100 nm.
FIG. 32 is an electron micrograph showing self-association of “laying” transthyretin-derived peptides. The scale bar represents 100 nm.
FIG. 33 is an electron micrograph showing self-association of “laying” lysozyme-derived peptides. The scale bar represents 100 nm.
FIG. 34 is an electron micrograph showing self-association of “laying” fibrinogen-derived peptides. The scale bar represents 100 nm.
FIG. 35 is an electron micrograph showing self-association of “laying” insulin-derived peptides. The scale bar represents 100 nm.
FIG. 36 is an electron micrograph showing self-association of “laying” prolactin-derived peptides. The scale bar represents 100 nm.
FIG. 37 is an electron micrograph showing self-association of “laying” β2 microglobulin derived peptides. The scale bar represents 100 nm.
FIG. 38 is a graph showing the effect of inhibitory peptides on IAPP self-association. Square-wild-type (wt) IAPP peptide; triangle-wt-IAPP + inhibitory peptide; circle-no peptide.
FIG. 39 is a graph showing the absorbance of light at 405 nm as a function of time during fibril formation, and thus the absorbance reflecting the aggregation kinetics of the IAPP-derived peptide (SEQ ID NOs: 46-49).
FIG. 40 shows a histogram showing the turbidity of the IAPP analog after aging for 7 days.
41a-f are electron micrographs of the “laying” IAPP analog. NFGAILSS-Figure 41a, NFGAILSS-Figure 41b, NIGAILSS-Figure 41c, NLGAILSS-Figure 41d, NVGAILSS-Figure 41e, and NAGAILSS-Figure 41f. The scale bar shown represents 100 nm.
Figures 42a-c show binding of IAPP-NFGAILSS to analogs of the minimal amyloidogenic sequence SNNXGAILSS (X = any natural amino acid except cysteine). FIG. 42a shows the short-term exposure of the bound peptide array. FIG. 42b shows long-term exposure of the bound peptide array. FIG. 42c shows quantification of short-term exposure (FIG. 42a) using densitometry and arbitrary units.
FIG. 43a shows possible configurations for all residues (yellow is fully possible, orange is partially possible), for L-proline (blue), and for the achiral Aib residue (magenta). Is a Ramachandran plot showing FIG. 43b-c shows the chemical structure of the long wild-type IAPP peptide (ANFLVH, SEQ ID NO: 124, FIG. 43b) and its Aib modified structure peptide (Aib-NF-Aib-VH, SEQ ID NO: 125, FIG. 43c). It is a schematic diagram shown. Functional groups suitable for modification are marked in blue (FIG. 43b), while modified groups are marked in red (FIG. 43c).
Figures 44a-d are electron micrographs of "laying" IAPP analogs. 44a-ANFLVH; 44b-ANFLV; 44c-Aib-NF-Aib-VH; 44d-Aib-NF-Aib-V. The scale bar shown represents 100 nm.
FIGS. 45a-d are photomicrographs showing Congo red binding to pre-associated wild type and Aib modified IAPP peptides. Polarized field micrographs are shown for each of the following laid (ie, day 11) peptide suspensions. 45a-ANFLVH; FIG. 45b-ANFLV; FIG. 45c-Aib-NF-Aib-VH; FIG. 45d-Aib-NF-Aib-V.
46a-b are graphs showing the secondary structure of insoluble wild type and Aib modified hIAPP aggregates as determined by Fourier Transform Infrared Spectroscopy (FI-IR). FIG. 46a—Wild type peptide ANFLVH and the corresponding Aib modified peptide as indicated by the arrow. FIG. 46b—Wild type ANFLVH and the corresponding Aib modified peptide as indicated by the arrow.
FIG. 47 is a graph showing the inhibitory effect of Aib-modified peptides on amyloid fibril formation. Wild type IAPP was incubated alone or with various peptides of the invention. Fibril formation as a function of time was determined using ThT fluorescence.
FIG. 48 is a histogram showing the inhibitory effect of short aromatic sequences (SEQ ID NOs: 112-123) on IAPP-self-association.
Figures 49a-d are graphs representing repeated cycles of selection of IAPP fibrillization inhibitors. Fibrosis was monitored by a ThT fluorescence assay. The fluorescence value of IAPP alone (4 μM) or the fluorescence value of IAPP in the presence of the assayed compound (40 μM) was tested. Measurements were made after IAPP fluorescence was achieved plateau. The IAPP fluorescence was arbitrarily set as 100. FIG. 49a shows the results of the first round of selection of IAPP fibrillation inhibitor. EG1 = D-Phe-D-Phe-D-Pro; EG2 = Aib-D-Phe-D-Asn-Aib; EG3 = D-Phe-D-Asn-D-Pro; EG4 = Aib-Asn-Phe- EG5 = Gln-Lys-Leu-Val-Phe-Phe; EG6 = Tyr-Tyr; EG7 = Tyr-Tyr-NH2; EG8 = Aib-Phe-Phe. FIG. 49b shows the results of the second round of selection of IAPP fibrillation inhibitor. EG13 = Asn-Tyr-Aib; EG14 = Asn-Tyr-Pro; EG15 = D-Pro-D-Tyr-D-Asn; EG16 = D-Tyr-Aib; EG17 = D-Pro-D-Tyr; EG18 = D-Tyr-D-Pro. FIG. 49c shows the results of the third round of selection of IAPP fibrillation inhibitor. d-F-P = D-Phe-Pro; P-dF = Pro-D-Phe; EG19 = Asn-Tyr-Tyr-Pro; EG20 = Tyr-Tyr-Aib; EG21 = Aib-Tyr-Tyr; EG22 = Aib-Tyr-Tyr-Aib; EG23 = D-Asn-Tyr-Tyr-D-Pro. FIG. 49d shows the results of the fourth round of selection of IAPP fibrillation inhibitor. EG24 = Pro-Tyr-Tyr; EG25 = Tyr-Tyr-Pro; EG26 = Pro-Tyr-Tyr-Pro; EG27 = D-Tyr-D-Tyr; EG28 = D-Pro-Aib; EG29 = D-Phe- EG30 = D-Trp-Aib; EG31 = D-Trp-D-Pro.
FIG. 50 is a graph showing inhibition of Aβ (1-40) fibril formation by D-Trp-Aib. Aβ1-40 stock solution is 10 mM D-Trp-Aib (triangle) in 100 mM NaCl, 10 mM sodium phosphate buffer (pH 7.4) to a final concentration of 5 μM, or any additive Diluted without (square). Fluorescence values were measured after adding 0.3 μM Tht to each sample. The result represents the average of two independent measurements.
FIGS. 51a-c are photomicrographs showing the inhibitory effect of D-Trp-Aib on Aβ fibrillation as visualized by TEM. FIG. 51a shows Aβ alone. FIGS. 51b-c show two different fields that were Aβ incubated in the presence of inhibitors.

  The present invention relates to novel peptides for diagnosing or treating amyloid-related diseases such as type II diabetes, antibodies thereto, compositions containing them, and methods of use of each.

  The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

  Before describing in detail at least one embodiment of the present invention, it is understood that the present invention is not limited in its application to the details of construction and arrangement of the components set forth in the following description or illustrated in the drawings. There must be. The invention is capable of other embodiments or of being practiced in various ways or being carried out in various ways. It should also be understood that the expressions and terms used herein are for purposes of illustration and should not be considered limiting.

  Numerous therapeutic methods for preventing amyloid fibril formation or degrading amyloid material have been described in the prior art. However, current treatment methods are limited by barriers such as cytotoxicity, non-specificity and delivery.

  While putting the invention into practice, and in search of a new treatment modality for amyloid-related diseases such as type II diabetes, the inventor has identified amyloidogenic peptides that cause fibril formation. A characteristic sequence was identified. This finding suggests that ordered amyloid formation involves a previously described mechanism involving non-specific hydrophobic interactions [Petkova (2002), Proc. Natl. Acad. Sci. USA, 99: 16742-16747], but rather suggests that a specific pattern of molecular interactions is involved.

  As illustrated herein below and further in the Examples section below, the inventor considered a very important role for aromatic residues in amyloid formation. The involvement of aromatic residues in the amyloid formation process is consistent with the well-established role of pi-stacking interactions in molecular recognition and self-association [Gillard et al. (1997) Chem. Eur. J. et al. 3: 1933-340; Claessens and Staudart (1997), J. MoI. Phys. Org. Chem. 10: 254-72; Shetty et al. (1996), J. MoI. Am. Chem. Soc. , 118: 1019-27; McGuaghey et al. (1998), π-stacking interactions: function in proteins. Biol. Chem. 273, 15458-15463; Sun and Bernstein (1996), J. MoI. Phys. Chem. 100: 13348-66]. π-stacking interactions are non-bonded interactions that are formed between planar aromatic rings. The steric constraints associated with the formation of such ordered stacking structures have a fundamental role in the self-association process that results in the formation of supramolecular structures. Such π-stacking interactions are probably entropy driven, but stabilize the double helix structure of DNA, core packing and stabilization of protein tertiary structure, host-guest interactions, and in solution. It plays a central role in many biological processes such as porphyrin aggregation [for a further review of the possible role of pi-stacking interactions in amyloid fibril self-association, see Gazit (2002), FASEB J. et al. 16: 77-83].

  The inventor will be able to create for the first time highly effective diagnostic, prophylactic and therapeutic peptides that can be used to treat or diagnose diseases characterized by the formation of amyloid plaques. The ability to mediate molecular recognition of short aromatic peptides such as dipeptides (see Examples 45-47) was demonstrated.

  Thus, according to one aspect of the present invention, there is provided a peptide comprising the amino acid sequence XY or YX, where X is an aromatic amino acid and Y is an amino acid other than glycine. Examples of peptides containing this sequence are set forth in SEQ ID NOs: 4, 12-19, 27-45, 112-123, 125 and 127. As shown by the results given in Examples 36-39 in the Examples section below, the inventor found that, contrary to the teachings of the prior art, it is rather than hydrophobic that it defines amyloid self-association. It was discovered to be aromatic. Therefore, the aromatic amino acids of the peptides of the present invention are very important for the formation of amyloid fibrils.

  An aromatic amino acid can be any naturally occurring aromatic residue or including phenylalanine, tyrosine, tryptophan, phenylglycine, or a modified, precursor or functional aromatic moiety thereof, or It may be a synthesized aromatic residue. Examples of aromatic residues that can be formed as part of the peptide of the present invention are shown in Table 2 below.

  As evidenced by the results presented in the Examples section below, the present invention facilitates the design of peptides that exhibit varying degrees of self-aggregation kinetics and aggregate structure.

  The expression “self-aggregation” as used herein refers to the ability of a peptide to form aggregates (eg, fibrils) in aqueous solution. The ability of a peptide to self-aggregate, and the kinetics and type of such self-aggregation, determine the use of the peptide in the treatment or diagnosis of amyloid disease.

  Since the aggregation kinetics and aggregate structure are mainly determined by the specific residue composition and possibly the length (see FIG. 1) of the peptide produced, the present invention is based on SEQ ID NOs: 4, 12-19, 27 Longer peptides (e.g. 10 amino acids to 50 amino acids) comprising the sequence shown in ~ 45, 112-123, 125, 127, 128-147 or 148, or preferably any of these sequences Shorter peptides (e.g. 2 to 15 amino acids, preferably at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, such as 12 amino acids, Preferably both of 15 amino acids or less).

  In order to increase the rate of amyloid formation, the peptides of the present invention preferably further comprise at least one amino acid having no polar charge, such as serine, threonine, asparagine, glutamine, or These natural derivatives or synthetic derivatives include, but are not limited to (see Table 2).

  According to one embodiment of this aspect of the invention, amino acid residue Y is an amino acid that has no polar charge.

  According to another embodiment of this aspect of the invention, the peptide has at least 3 amino acids, an XY / YX amino acid sequence as described above and upstream of the XY / YX sequence (N- Include amino acids with no additional polar charge located either terminally or downstream (C-terminal).

  The peptides of the invention can be at least 3 amino acids in length and have at least one pair of positively charged amino acids (eg lysine and arginine) and negatively charged amino acids (eg Aspartic acid and glutamic acid) (eg, SEQ ID NOs: 27-29). Such an amino acid composition may be preferred. This is because, as shown in Example 21 of the Examples section, electrostatic interactions between opposite charges can induce the formation of an ordered antiparallel structure.

  Furthermore, the peptides of the invention can be 4 amino acids long and contain two serine residues at the C-terminus of the XY / YX sequence.

Further, the peptides of the invention can be 3 amino acids long and contain a thiolated amino acid residue (ie, containing a sulfur ion), preferably at its N-terminus (eg, SEQ ID NO: 149). And 150, D-Cys-D-Trp-Aib and L-Cys-D-Trp-Aib, respectively, and their acylated and amidated forms). Such peptide forms are very valuable. Because it imparts reducing properties to the peptide, thereby as a reducing agent and antioxidant (both can be important for neuroprotection (Offen et al. (2004), J Neurochem. 89: 1241-51); and Can act both as amyloid inhibitors Examples of thiolated amino acids include, but are not limited to, the naturally occurring amino acids cysteine and methionine and synthetic amino acids such as Tyr (SO ₃ H).

  Since we have identified the sequence features that govern fibril formation, the teachings of the present invention also do not agglomerate into fibrils, preventing or reducing fibril formation, or previously Either breaking the formed fibrils is possible, thus allowing the design of peptides that can be used as therapeutic agents.

  For example, the peptides encompassed by SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 25 or SEQ ID NO: 30 can be utilized for therapy. This is because, as shown in the Examples section below, such peptides show no aggregation (SEQ ID NO: 9) or slow aggregation kinetics when compared to the wild type peptide ( From SEQ ID NO: 9 and SEQ ID NO: 10). Since amyloid formation is a very slow process, these peptide sequences are believed to completely inhibit or significantly delay amyloidosis under physiological conditions.

As used herein, the term “peptide” refers to a natural peptide (either a degradation product, a synthetic peptide, or a recombinant peptide) as well as a peptide that has been modified to be more stable in the living body or more permeable into cells. Peptide mimetics (typically synthetic peptides) such as peptoids and semipeptoids, which may be peptide analogs (peptide analogs). Such modifications include, but are not limited to, N-terminal modification, C-terminal modification, peptide bond modification, CH ₂ —NH, CH ₂ —S, CH ₂ —S═O, O═C—NH. , CH ₂ —O, CH ₂ —CH ₂ , S═C—NH, CH═CH or CF═CH, skeletal modifications and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are described, for example, in Quantitative Drug Design, C.I. A. Ramsden Gd. , Chapter 17.2, F. Specified in Choplin Pergamon publication (1992), which is incorporated by reference as if fully set forth herein. Further details on this are described below.

A peptide bond (—CO—NH—) in a peptide can be converted into, for example, an N-methylated bond (—N (CH ₃ ) —CO—), an ester bond (—C (R) H—C—O—O—C ( R) —N—), ketomethylene bond (—CO—CH ₂ —), α-aza bond (—NH—N (R) —CO—) (wherein R is an alkyl group such as methyl), carba bond (—CH ₂ —NH—), hydroxyethylene bond (—CH (OH) —CH ₂ —), thioamide bond (—CS—NH—), olefin double bond (—CH═CH—), retroamide bond ( -NH-CO-), peptide derivatives _{(-N (R) -CH 2 -CO} -) ( wherein, R may be substituted by the "normal" side chain) that are naturally present on the carbon atom.

  These modifications may occur at any of the bonds along the peptide chain, and may occur at several places (2 to 3 places) at the same time.

  Synthesis of natural aromatic amino acids, Trp, Tyr and Phe, such as phenylglycine, Tic, naphthylalanine (Nal), phenylisoserine, threoninol, cyclic methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr It may be substituted with a non-natural acid.

  In addition to the above, the peptides of the present invention may also comprise one or more modified amino acids or one or more non-amino acid monomers (eg fatty acids, carbohydrate complexes, etc.).

  The term “amino acid” or “amino acid (s)” as used herein and in the claims section refers to the 20 naturally occurring amino acids that are often post-translationally modified in vivo. Other unusual amino acids such as, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine, including, for example, hydroxyproline, phosphoserine and phosphothreonine. It should be understood as including. Furthermore, the term “amino acid” includes both D- and L-amino acids.

Tables 1 and 2 below list naturally occurring amino acids (Table 1) and non-conventional or modified amino acids (Table 2) used in the present invention.

  Since the peptides of the present invention are preferably utilized in therapeutics or diagnostics where the peptide is required to be in a soluble form, the peptides of the present invention are preferably one or more non-natural or natural Types of polar amino acids, such amino acids include, but are not limited to, serine and threonine, which can increase the solubility of the peptide through its hydroxyl-containing side chains.

  For therapeutic purposes, the peptides of the present invention preferably comprise at least one β-sheet breaker amino acid residue, which is located in a peptide sequence as described below. Peptides containing such β-sheet disruptor amino acids retain amyloid polypeptide recognition but prevent its aggregation (see Examples 40-45 in the Examples section below). According to one preferred embodiment of this aspect of the invention, the β-sheet breaker amino acid is about −60 degrees to +25 degrees rather than the typical β-sheet φ angle of about −120 degrees to −140 degrees. Of naturally occurring amino acids such as proline (eg, SEQ ID NOs: 45, 112, 119, 120, 122, etc.), which are characterized by a limited φ angle, thereby destroying the β-sheet structure of amyloid fibrils. 123, 128, 130, 134, 138, 139, 140, 141, 143, 144, 146, 147 and 148, see section "Background Art"). Other β-sheet breaker amino acids include, but are not limited to, aspartic acid, glutamic acid, glycine, lysine and serine (according to Chou and Fasman (1978), Annu. Rev. Biochem. 47,258).

  According to another preferred embodiment of this aspect of the invention, the β-sheet breaker amino acid residue is a synthetic amino acid, such as a Cα-methylated amino acid, and its conformational conditions are limited [Balaram, (1999). ), J .; Pept. Res. 54, 195-199]. Unlike natural amino acids, Cα-methylated amino acids have a hydrogen atom bonded to Cα, which has a wide influence on their steric properties with respect to the φ and ψ angles of amide bonds. Thus, alanine has a wide range of possible φ and ψ configurations, while α-aminoisobutyric acid (Aib, see Table 2 above) has a limited φ and ψ configuration. Thus, peptides of the invention that are substituted with at least one Aib residue can bind amyloid polypeptides but prevent their aggregation (see Examples 40-44). Such peptides are described in SEQ ID NOs: 113, 114, 117, 118, 121, 135, 136, 137, 143, 145, 149, 129 and 131.

  The β-sheet disrupter amino acid of this aspect of the invention can be located at position Y of the XY / YX amino acid sequence of the peptide (eg, SEQ ID NOs: 123, 143, 144, 145, 146, 147). 148). Alternatively, the peptide of this aspect of the invention can be at least 3 amino acids and includes a disrupter amino acid at any position other than the XY / YX amino acid sequence (see, eg, SEQ ID NO: 117). .

  The β-sheet breaker amino acid may be located upstream of the aromatic residue (see SEQ ID NO: 122) or downstream (see SEQ ID NO: 123).

  According to one preferred embodiment of this aspect of the invention, the peptide is 3 amino acids long, Y is an aromatic amino acid, and the amino acid residue bound to the amino acid sequence XY or Y-X. The group is a β-sheet breaker amino acid, which is preferably attached at the C-terminus of the peptide (eg, SEQ ID NO: 135 and 140).

  According to another preferred embodiment of this aspect of the invention, the peptide is 2 amino acids in length and Y is a β-sheet breaker amino acid (eg, SEQ ID NOs: 121, 143-148).

According to the most preferred embodiment of this aspect of the invention, the peptide is a dipeptide having the general formula:
Where C ^* is a chiral carbon having the D configuration (also referred to as the R configuration)
R ₁ and R ₂ are each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carboxy, thiocarboxy, C-carboxylate and C-thiocarboxylate;
R ₃ is selected from the group consisting of hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, halo and amine; and R ₄ is alkyl.

  The term “alkyl” as used herein refers to a saturated aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 1 to 20 carbon atoms. Whenever a numerical range, such as “1-20” is mentioned herein, it means that the group (in this case an alkyl group) is 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. Of up to 20 carbon atoms. More preferably, the alkyl group is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkyl group is a lower alkyl having 1 to 4 carbon atoms. Alkyl groups can be substituted or unsubstituted. When substituted, the substituents can be, for example, halo, hydroxy, cyano, nitro and amino.

  A “cycloalkyl” group refers to a monocyclic or fused ring (ie, ring that shares a pair of adjacent carbon atoms) group consisting of all carbons, in which one or more of the rings do not have a fully conjugated pi-electron system. Non-limiting examples of cycloalkyl groups include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene and adamantane. Cycloalkyl groups can be substituted or unsubstituted. When substituted, the substituents can be, for example, alkyl, halo, hydroxy, cyano, nitro and amino.

  An “aryl” group refers to a monocyclic or fused polycyclic (ie, ring that shares a pair of adjacent carbon atoms) group consisting of all carbons with a fully conjugated pi-electron system. Non-limiting examples of aryl groups include phenyl, naphthalenyl and anthracenyl. Aryl groups can be substituted or unsubstituted. When substituted, the substituents can be, for example, alkyl, cycloalkyl, halo, hydroxy, alkoxy, thiohydroxy, thioalkoxy, cyano, nitro and amino.

  A “hydroxy” group refers to an —OH group.

  An “alkoxy” group refers to both an —O-alkyl and an —O-cycloalkyl group, as defined herein.

  An “aryloxy” group refers to an —O-aryl group, as defined herein.

  A “thiohydroxy” group refers to a —SH group.

  A “thioalkoxy” group refers to both an —S-alkyl and an —S-cycloalkyl group, as defined herein.

  A “thioaryloxy” group refers to an —S-aryl group, as defined herein.

  A “carboxy” group is a —C (═O) —R ′ group, where R ′ represents hydrogen, halo, alkyl, cycloalkyl or aryl, as defined herein.

  A “thiocarboxy” group refers to a —C (═S) —R ′ group, where R ′ is as defined herein.

  A “C-carboxylate” group refers to a —C (═O) —O—R ′ group, where R ′ is as defined herein.

  An “O-thiocarboxylate” group refers to a —C (═S) —O—R ′ group, where R ′ is as defined herein.

  A “halo” group refers to fluorine, chlorine, bromine or iodine.

  An “amine” group refers to a —NR′R ″ group, where R ′ is as defined herein and R ″ is as defined for R ′.

A “nitro” group refers to a —NO ₂ group.

  A “cyano” group refers to a —C≡N group.

Preferably, R ₄ is methyl, so the above compound is D-tryptophan-α-aminobutyric acid (also referred to as D-Trp-aib or D-tryptophan-α-methyl-alanine), or a derivative thereof. is there.

  An unmodified dipeptide, a peptide having an L-configuration, a peptide having an inverted configuration (ie, C-N sequence of tryptophan (D / L) and α-methylalanine), or a macromolecule comprising the peptide sequence It will be appreciated that (eg, peptides, immobilized peptides) are known (eg, WO 02/094857, WO 02/094857, EP Patent No. 966975, US Patent No. 6255286, 6215625, 6162828). And 5304470). However, such molecules are chemically and biologically different from the peptides described above and their unique activity depends strictly on their structure.

  The peptides of the present invention are preferably utilized in a linear form, but it is understood that cyclic forms of peptides can also be utilized if cyclization does not significantly interfere with peptide properties.

  Cyclic peptides can either be synthesized in a cyclic form or can be configured to take a cyclic form under desired conditions (eg, physiological conditions).

For example, a peptide according to the teachings of the present invention can include at least two cysteine residues adjacent to the core peptide sequence. In this case, the cyclization can be created by the formation of an SS bond between the two Cys residues. Side chain to side chain cyclization may also be created by the formation of an interactive bond of the formula — (— CH ₂ —) _n —S—CH ₂ —C—, where n = 1 or 2. This is possible, for example, by inclusion of Cys or homoCys and reaction of its free SH group with eg bromoacetylated Lys, Orn, Dab or Dap. In addition, cyclization can be accomplished, for example, by amide bond formation, for example, Glu, Asp, Lys, Orn, diaminobutyl (Dab) acid, diaminopropion (Dap) acid in various chains (—CO—NH or —NH—CO bonds). It can be obtained by including it in various positions. Cyclization of skeleton and skeletal also has the formula _{H-N ((CH 2)} n -COOH) -C (R) H-COOH or _{H-N ((CH 2)} n -COOH) -C (R) H- It can be obtained with a modified amino acid of NH ₂ , where n = 1 to 4 and R is a natural or non-natural side chain of the amino acid.

  The present invention thus provides conclusive data regarding the identity of structural determinants of amyloid peptides that lead to fibril assembly.

  Thus, the present invention allows the design of various peptide sequences that can be utilized for the prevention / treatment or diagnosis of amyloidosis.

  As described in Examples 6-35, it is understood that the inventors were able to identify the consensus aromatic sequence of the present invention (SEQ ID NO: 7) in a number of amyloid-related proteins. Thus, the present invention makes it possible to accurately identify amyloidogenic fragments of essentially all amyloidogenic proteins.

  In addition, small aromatic molecules such as Ro47-1816 / 001 [Kuner et al. (2000) J. MoI. Biol. Chem. 275: 1673-8, see FIG. 2a] and 3-p-toluoyl-2- [4 ′-(3-diethylaminopropoxy) phenyl] benzofuran [Twyman (1999), Tetrahedron Letters, 40: 9383-9384] and the like. Have been shown to be effective in inhibiting the polymerization of β-polypeptides of Alzheimer's disease [Findeis et al. (2000), Biochem. Biophys. Acta, 1503: 76-84], on the other hand, amyloid-specific dyes containing aromatic elements such as Congo Red (FIG. 2b) and Thioflavin T (FIG. 2c) are common amyloid formation inhibitors. This fact demonstrates that the recognition motif of the present invention is sufficient for amyloid self-association.

  The ability to obtain the peptides of the present invention makes it possible to produce antibodies against the peptides of the present invention that can be used to dissociate amyloid plaques or prevent the formation of amyloid plaques ( US Pat. No. 5,688,561).

The term “antibody” refers to intact antibody molecules and functional fragments thereof that are capable of binding to macrophages, such as Fab, F (ab ′) ₂ and Fv. These functional antibody fragments are defined as follows: (i) Fab, a fragment containing a monovalent antigen-binding fragment of an antibody molecule and digesting the complete antibody with the enzyme papain Fragment that can be produced by generating a light chain and part of one heavy chain; (ii) Fab ′, a complete antibody treated with pepsin and then reduced to form a complete light chain A fragment of an antibody molecule that can be obtained by generating part of a heavy chain; two Fab ′ fragments are obtained per antibody molecule; (iii) F (ab ′) ₂ , a complete antibody, without subsequent reduction, fragment of an antibody that can be obtained by treating with the enzyme pepsin; F (ab ') _2, the two Fab' fragments of two Ges Dimers linked together by fido bonds; (iv) Fv, which is defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains And (v) a genetically engineered containing a light chain variable region and a heavy chain variable region linked by a polypeptide linker suitable as a single chain antibody ("SCA"), a gene fused single chain molecule. Molecules.

  Various methods of making these fragments are known in the art (see, eg, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, which is incorporated by reference). about).

  Various methods for making antibodies (ie, monoclonal and polyclonal) are well known in the art. Antibodies can be made by any one of several methods known in the art. In such methods, induction of in vivo production of antibody molecules, screening a library of immunoglobulin libraries or highly specific binding reagents as disclosed [Orlandi D. et al. R. Et al. (1989), Proc. Natl. Acad. Sci. 86: 3833-3837; Winter G. et al. Et al. (1991), Nature, 349: 293-299], or the production of monoclonal antibody molecules by continuous cell lines in culture can be used. These include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and Epstein-Barr virus (EBV) -hybridoma technology [Kohler G. et al. Et al. (1975) Nature 256: 495-497; Kozbor D. et al. Et al. (1985), J. Am. Immunol. Methods, 81: 31-42; J. et al. Et al. (1983), Proc. Natl. Acad. Sci. 80: 2026-2030; Cole S .; P. Et al. (1984), Mol. Cell. Biol. 62: 109-120].

  Antibody fragments according to the present invention are prepared by proteolytic hydrolysis of the antibody or by expression of the DNA encoding the fragment in E. coli or mammalian cells (eg, Chinese hamster ovary cell culture or other protein expression systems). be able to.

Antibody fragments can be obtained by pepsin digestion or papain digestion of complete antibodies by conventional methods. For example, antibody fragments can be generated by enzymatic cleavage of antibodies with pepsin to obtain a 5S fragment denoted as F (ab ′) ₂ . This fragment is further cleaved using a thiol reducing agent and, if necessary, a protecting group for the sulfhydryl group resulting from cleavage of the disulfide bond to create a 3.5S Fab ′ monovalent fragment. be able to. Alternatively, enzymatic cleavage using pepsin produces two monovalent Fab ′ fragments and one Fc fragment directly. These methods are described, for example, in Goldenberg, US Pat. Nos. 4,036,945 and 4,331,647, and references contained therein (such patents are hereby incorporated by reference in their entirety). . Porter, R.A. R. Biochem. J. et al. 73: 119-126, 1959. As long as the fragment binds to the antigen recognized by the intact antibody, other methods of cleaving the antibody, such as separation of the heavy chain to form a monovalent light-heavy chain fragment, further cleavage of the fragment, or Other enzymatic, chemical or genetic techniques can also be used.

Fv fragments comprise an association of V _H and V _L chains. This meeting was organized by Inbar et al., Proc. Nat'l. Acad. Sci. USA, 69: 2659-62, 1972, which may be non-covalent. Alternatively, the variable chains can be linked by intermolecular disulfide bonds or can be crosslinked by chemical reagents such as glutaraldehyde. Preferably, the Fv fragment comprises a V _H and V _L chain connected by a peptide linker. These single chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the _VH and _VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is then introduced into a host cell such as E. coli. Recombinant host cells synthesize a single peptide chain in which the linker peptide connects the two V domains. Various methods for producing sFv are described in, for example, Whitlow and Filpula Methods, 2: 97-105, 1991; Bird et al., Science, 242: 423-426, 1988; Pack et al., Bio / Technology, 11: 1271. ˜77, 1993; Ladner et al., US Pat. No. 4,946,778, which are hereby incorporated by reference in their entirety.

  Another form of antibody fragment is a peptide that encodes only one complementarity determining region (CDR). CDR peptides (“minimal recognition units”) can be obtained by constructing a gene encoding the CDR of the antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize variable regions from the RNA of antibody producing cells. See, for example, Larrick and Fry, Methods 2: 106-10, 1991.

For human application, the antibodies of the present invention are preferably humanized. Humanized forms of non-human (eg, murine) antibodies are immunoglobulins, immunoglobulin chains or fragments thereof (eg, Fv, Fab, Fab ′, F () that contain minimal sequence derived from non-human immunoglobulin. ab ′) ₂ , or other antigen-binding subsequences of antibodies)). Humanized antibodies include residues from non-human species (donor antibodies), such as mice, rats or rabbits, in which residues from the recipient's complementary determining region (CDR) have the desired specificity, affinity and ability. Human immunoglobulins (recipient antibodies) that are replaced by residues derived from CDRs are included. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues that are found neither in the recipient antibody nor in the introduced CDR or framework sequences. In general, a humanized antibody comprises substantially all variable domains, or at least one variable domain, typically two variable domains, wherein all or substantially all of the CDR regions are non-human immunoglobulin. And all or substantially all of the FR regions are FR regions of a human immunoglobulin consensus sequence. Humanized antibodies also optimally comprise at least a portion of an immunoglobulin constant region (Fc), and typically comprise at least a portion of the constant region of a human immunoglobulin [Jones et al., Nature, 321: 522. -525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992)].

  Various methods for humanizing non-human antibodies are widely known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often shown as introduced residues that are typically selected from introduced variable domains. Humanization essentially consists of the method of Winter and co-workers [Jones et al., Nature, 321: 522-525 (1986) by replacing rodent CDRs or CDR sequences with the corresponding sequences of human antibodies. Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)]. Accordingly, such humanized antibodies are chimeric antibodies in which substantially but not all human variable domains are replaced by corresponding sequences from non-human species (US Pat. No. 4,816,567). In fact, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

  Human antibodies are also available from phage display libraries [Hoogenboom and Winter, J. MoI. Mol. Biol. 227: 381 (1991); Marks et al., J. MoI. Mol. Biol. 222: 581 (1991)], and can be manufactured using a variety of techniques known in the art. The techniques of Cole et al. And Boerner et al. Can also be utilized to prepare human monoclonal antibodies [Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R., et al. Liss, p. 77 (1985); Boerner et al. Immunol. 147 (1): 86-95 (1991)]. Similarly, human antibodies can be generated by introducing human immunoglobulin loci into transgenic animals (eg, mice) in which endogenous immunoglobulin genes are partially or completely inactivated. it can. Upon antigen stimulation, human antibody production is observed, which is very similar to antibody production seen in humans in everything including gene rearrangement, assembly and antibody repertoire. This method is described, for example, in US Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, and described in the following scientific publications. : Marks et al., Bio / Technology, 10, 779-783 (1992); Lonberg et al., Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812-13 (1994); Fishwild et al., Nature. Biotechnology, 14, 845-51 (1996); Neuberger, Nature Biotechnology, 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

  As mentioned hereinabove, one specific use for the peptides of the invention is the prevention or treatment of diseases associated with amyloid plaque formation.

  Thus, according to yet another aspect of the present invention, a method for treating an amyloid-related disease in an individual is provided. Preferred individual subjects according to the present invention are mammals, such as dogs, cats, goats, pigs, horses, cows, humans and the like.

  The term “treating” refers to reducing or preventing amyloid plaque formation or substantially reducing the appearance of plaques in affected tissues. The expression “amyloid plaques” refers to fibrillar amyloid, as well as aggregated but not fibrillar amyloid (hereinafter “protofibrillar amyloid”, which can also be pathogenic). For example, an aggregated but not necessarily fibrillar form of IAPP was found to be toxic in culture. As demonstrated by Anaguiano and co-workers [(2002), Biochemistry, 41: 11338-43], protofibrillar IAPP is similar to protofibrillar α-sinucerin, which is implicated in the pathogenesis of Parkinson's disease, The synthesized vesicles were permeated by a pore-like mechanism. The formation of IAPP amyloid pores was temporarily correlated with the early formation and loss of IAPP oligomers to the appearance of amyloid fibrils. These results suggest that protofibrillar IAPP may be as important for type II diabetes as other protofibrillar proteins are important for the development of Alzheimer's and Parkinson's disease.

  Amyloid-related diseases to be treated according to the present invention include type II diabetes, Alzheimer's disease (AD), early-onset Alzheimer's disease, late-onset Alzheimer's disease, prearranged Alzheimer's disease, Parkinson's disease, SAA amyloidosis, hereditary Iceland syndrome , Multiple myeloma, medullary cancer, medical carcinoma of the aorta, insulin injection amyloidosis, prion systemic amyloidosis, chronic inflammatory amyloidosis, Huntington's disease, senile systemic amyloidosis, pituitary amyloidosis, hereditary renal amyloidosis, familial British dementia, Finnish hereditary amyloidosis, familial non-neuropathic amyloidosis [Gazit (2002), Curr. Med. Chem. 9: 1667-1675] and prion diseases (including sheep and goat scrapie and bovine bovine spongiform encephalopathy (BSE)) [Wilesmith and Wells (1991), Curr. Top. Microbiol. Immunol. 172: 21-38] and human prion disease (including (i) Kuru, (ii) Kreuzfeld-Jakob disease (CJD), (iii) Gerstmann-Streisler-Scheinker disease (GSS)) And (iv) fatal familial insomnia (FFI) is included] [Gajdusek (1977), Science, 197: 943-960; Medori, Tritschler et al. (1992), N Engl J Med, 326: 444-449] Is included, but is not limited thereto.

  The methods of the invention comprise providing a therapeutically effective amount of a peptide of the invention to an individual. The peptide can be provided using any one of a variety of delivery methods. Various delivery methods and suitable formulations are described hereinbelow with respect to pharmaceutical compositions.

  When used to treat amyloid disease, the peptides of the present invention may prevent fibril formation or reduce fibril formation, or compete with preformed aggregates By destabilizing, it is understood to include an amino acid sequence suitable for deaggregating the formed aggregate. For example, SEQ ID NOs: 45, 112-123, 125, 127, 128-149 and 150 can be utilized to treat amyloid diseases (particularly type II diabetes). This is evident by the low ability of amyloidogenic peptides to bind thioflavin T in the presence of inhibitory peptides, as shown in Examples 35 and 45 in the Examples section below. This is because it interferes with IAPP self-association.

  Alternatively, the peptide shown in SEQ ID NO: 10 or SEQ ID NO: 11 is induced by very slow aggregation kinetics due to substitution of either leucine or isoleucine in the peptide, as shown in the Examples section below. It can be used as a potent inhibitor of type 2 diabetes. Since amyloid formation in vivo is a very slow process, under physiological conditions, it is believed that fibrillation does not occur when isoleucine or leucine is replaced with alanine in the context of full-length IAPP.

  Alternatively, self-aggregating peptides such as those shown in SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO: 28 to SEQ ID NO: 30 can be used as potent inhibitors of amyloid fibrillation. This is because such peptides can form heteromolecular complexes that are not as ordered as homomolecular aggregates formed by amyloid fragments.

  Since one of the major obstacles in the use of short peptide fragments in therapy is its proteolytic degradation by stereospecific cellular proteases, the peptides of the present invention are preferably derived from D-isomers of natural amino acids. It is understood that they are synthesized [ie, inverso peptide analogs, Tjerberg (1997), J. MoI. Biol. Chem. 272: 12601-5; Gazit (2002), Curr. Med. Chem. 9: 1667-1675].

  Furthermore, the peptides of the present invention include their retro analogs, inverso analogs and retro-inverso analogs. It is understood that full retro-inverso analogs or extended partial retro-inverso analogs of hormones are generally found to retain or enhance biological activity. Retroinversion has also been applied in the field of rational design of enzyme inhibitors (see US Pat. No. 6,261,569).

  As used herein, “retropeptide” refers to a peptide composed of L-amino acid residues assembled in the opposite direction to the original peptide sequence.

  In retroinverso modifications of naturally occurring polypeptides, the α-carbon stereochemistry is from the amino acid opposite to the α-carbon stereochemistry of the corresponding L-amino acid, ie, from D-amino acids or D-allo-amino acids. , With synthetic assembly in the reverse order of the original peptide sequence. Thus, retroinverso analogs have reversed ends and reverse peptide bonds, while essentially maintaining the side chain topology as in the original peptide sequence.

  Furthermore, since one of the major issues in amyloid fibril formation is the transfer of amyloid polypeptide from its original form to a stacked β chain structure, inhibitory peptides preferably have a peptide backbone due to steric effects. Constraining N-methylated amino acids are included [Kapuriotu (2002), 315: 339-350]. For example, aminoisobutyric acid (Aib or methylalanine) is known to stabilize the α-helix structure in short natural peptides. Furthermore, N-methylation also affects the intermolecular NH-to-CO H-binding ability and thus suppresses the formation of multilayer β-chains that are stabilized by interactions that form H-bonds.

  Adding an organic group such as a cholyl group to the N-terminus or C-terminus of the peptides of the invention improves the efficacy and bioavailability of the therapeutic peptide (eg, crossing the blood brain barrier in the case of neurodegenerative diseases) Is preferred because it is shown [Findeis (1999), Biochemistry, 38: 6791-6800]. In addition, by introducing charged amino acids into the recognition motif, electrostatic repulsion can be generated that inhibits further growth of amyloid fibrils [Lowe (2001), J. Biol. Mol. Biol. 40: 7882-7889].

  As stated hereinabove, the antibodies of the invention can also be used to treat amyloid-related diseases.

  The peptides and / or antibodies of the invention can be given to an individual as such or as part of a pharmaceutical composition mixed with a pharmaceutically acceptable carrier.

  As used herein, “pharmaceutical composition” refers to a formulation of one or more of the active ingredients described herein with other chemical ingredients (eg, physiologically suitable carriers and excipients). . The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

  As used herein, the term “active ingredient” refers to a peptide or antibody preparation that can explain a biological effect.

  Hereinafter, the terms “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” used interchangeably do not cause significant irritation to the organism and inhibit the biological activity and properties of the administered compound. Does not refer to a carrier or diluent. An adjuvant is included in these terms. One of the components contained in a pharmaceutically acceptable carrier can be, for example, polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media [Mutter et al. (1979)].

  As used herein, the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, various types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

  Techniques relating to drug formulation and administration are described in “Remington's Pharmaceutical Sciences”, Mack Publishing Co. (Easton, PA) can be found in the latest edition, which is incorporated herein by reference.

  Suitable administration routes include, for example, oral delivery, rectal delivery, transmucosal delivery, particularly nasal delivery, intestinal delivery or extraintestinal delivery (intramuscular, subcutaneous, and intramedullary, as well as intrathecal, direct brain Indoor injection, intravenous injection, intraperitoneal injection, intranasal injection, or intraocular injection).

  Rather than systemic methods, the preparations may also be administered in a local manner, such as by injecting the preparation directly into a specific area of the patient's body.

  The pharmaceutical composition of the present invention is manufactured by a method well known in the art, for example, using ordinary mixing, dissolution, granulation, sugar-coated tablet production, kneading, emulsification, encapsulation, capture or freeze-drying method, etc. Can do.

  The pharmaceutical compositions used in the present invention are prepared by conventional methods using one or more physiologically acceptable carriers containing excipients and adjuvants that facilitate the processing of the active ingredient into a pharmaceutically acceptable formulation. It can be formulated. Proper formulation is dependent upon the route of administration chosen.

  In the case of injection, the active ingredient of the present invention can be formulated in an aqueous solution (preferably a biocompatible buffer such as Hanks' solution, Ringer's solution, or physiological saline buffer). For transmucosal administration, penetrants appropriate to the wall to be permeated are added to the formulation. Such penetrants are widely known in the art.

  For oral administration, the compounds can be formulated readily by mixing the active compound with pharmaceutically acceptable carriers well known in the art. By using the carrier, the compound of the present invention can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for ingestion by patients. it can. Oral drugs use solid excipients, optionally pulverize the resulting mixture, add appropriate adjuvants if desired, and then process the granule mixture to form a tablet or sugar-coated tablet Can be manufactured. Suitable excipients include fillers such as lactose, sucrose, mannitol or sorbitol containing sugars, cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose Physiologically acceptable polymers such as sodium carbomethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof (eg, sodium alginate).

  Dragee cores are provided with suitable coatings. A concentrated sugar solution can be used for this purpose, which can be selected from gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solution, and a suitable organic solvent or A solvent mixture can be included. Dyestuffs or pigments may be added to the tablets or dragee tablet skins for identification or to characterize different combinations of active ingredient amounts.

  Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Indented capsules can contain active ingredients mixed with fillers such as lactose, binders such as starch, lubricants such as talc or magnesium stearate, and optionally stabilizers. In the case of soft capsules, the active ingredient can be dissolved or suspended in a suitable liquid (such as fatty oil, liquid paraffin or liquid polyethylene glycol). Further stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

  For oral mucosal administration, the composition can take the form of tablets or lozenges formulated by conventional methods.

  For administration by nasal inhalation, the active ingredient for use in the present invention is a pressurized container or nebulizer using a suitable propellant (eg, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide). Conveniently delivered in the form of an aerosol spray. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Dispenser (eg, gelatin) capsules and cartridges containing a powder mixture of the compound and a suitable powder base (such as lactose or starch) can also be formulated.

  The preparations described herein can also be formulated for parenteral administration, eg, by bolus injection or continuous infusion. The preparation for injection can be provided, for example, in a single-dose form in an ampoule or the like, or in a multi-dose container, with a preservative added as desired. The composition can be a suspension, solution or emulsion in an oily or aqueous vehicle and may contain pharmaceutical agents such as suspending, stabilizing and / or dispersing agents.

  Pharmaceutical compositions for parenteral administration include aqueous solutions of water-soluble active preparations. Suspensions of the active ingredients can also be prepared as appropriate oily or aqueous injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. If desired, the suspension may further comprise suitable stabilizers or compounds which increase the solubility of the active ingredient so that a highly concentrated solution preparation can be obtained.

  The active ingredient may also take the form of a powder composed of a suitable vehicle (eg, sterile pyrogen-free water) prior to use.

  The preparations of the invention can also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases (such as cocoa butter or other glycerides).

  Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredient is included in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredient effective to prevent, alleviate or ameliorate symptoms of disease or to prolong the survival of the subject being treated.

  Determination of a therapeutically effective amount can be within the ability of those skilled in the art.

  For preparations used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro assays. For example, doses can be formulated in animal models, and such information can be used to more accurately determine effective doses in humans.

  Toxicity and therapeutic efficacy of the active ingredients described herein can be determined in cell cultures or experimental animals by standard in vitro pharmaceutical techniques. The data obtained from these in vitro, cell culture assays and animal experiments can be used to develop dose ranges for use in humans. The dosage may vary within this range depending on the route of administration utilized. Individual physicians can select the exact formulation, route of administration, and dosage taking into account the patient's condition (see, eg, “The Pharmaceuticals of Therapeutics” (1975) Chapter 1, page 1 of Fing et al. See).

  Depending on the severity and responsiveness of the condition to be treated, the administration can be a single dose or multiple doses. In this case, treatment lasts for days to weeks or until healing is achieved or reduction of the disease state is achieved.

  The amount of composition to be administered will, of course, depend on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and the like.

  In addition, a composition comprising a preparation of the invention formulated in a compatible pharmaceutical carrier can be manufactured, placed in a suitable container, and labeled for treatment of the indicated condition.

  The compositions of the present invention can optionally be provided as a pack or dispenser device, eg, an FDA approved kit, containing one or more unit dosage forms containing the active ingredients. The pack may consist of a metal foil or a plastic foil, for example a blister pack. The pack or dispenser device may be accompanied by precautions for administration. The pack or dispenser carries a notice indicating the form of the composition or regulatory approval for administration to humans or animals, attached to the container in a form specified by a government agency that regulates the manufacture, use or sale of the drug. May be. Such notices can be, for example, labeling approved by the US Food and Drug Administration for prescription drugs, or approved attachments.

  It will be appreciated that the peptides or antibodies of the invention can also be expressed from nucleic acid constructs administered to an individual using any suitable mode of administration described hereinabove (ie, in vivo gene therapy). ). Alternatively, the nucleic acid construct is introduced into a suitable cell by an appropriate gene delivery vehicle / method (transfection, transduction, homologous recombination, etc.) and an expression system if required, after which the modified cell is cultured. Expanded and returned to the individual (ie, ex vivo gene therapy).

  In order to allow cellular expression of the peptides or antibodies of the invention, the nucleic acid construct of the invention further comprises at least one cis-acting regulatory element. The expression “cis-acting regulatory element” as used herein refers to a polynucleotide sequence (preferably a promoter) that binds to a trans-acting regulatory element and regulates the transcription of a coding sequence located downstream thereof.

  Any available promoter can be used by the methodology of the present invention. In a preferred embodiment of the invention, the promoter utilized by the nucleic acid construct of the invention is active in the particular cell population transformed. Examples of cell type specific and / or tissue specific promoters include promoters such as albumin that are liver specific [Pinkert et al. (1987), Genes Dev. 1: 268-277], lymphoid specific promoters [Calam et al. (1988), Adv. Immunol. 43: 235-275], in particular the T cell receptor promoter [Winoto et al. (1989), EMBO J. et al. 8: 729-733] and immunoglobulin promoters [Banerji et al. (1983) Cell, 33729-740], neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1986), Proc. Natl. Acad. Sci. USA, 86: 5473-5477], such as pancreas-specific promoters [Edrunch et al. (1985), Science, 230: 912-916], or mammary gland specific promoters such as whey promoters (US Pat. No. 4,873,316 and European patents). Application Publication No. 264166) and the like are included. The nucleic acid constructs of the present invention can further comprise an enhancer, which can be present near or far from the promoter sequence and can function to up regulate transcription from the promoter.

  The methodology constructs of the present invention preferably further include an appropriate selectable marker and / or origin of replication. Preferably, the construct utilized is a shuttle vector, which can be propagated together in E. coli (in which case the construct contains a suitable selectable marker and origin of replication), the growth in the cell, the selected gene and Can be compatible for incorporation in tissues. The construct according to the invention can be, for example, a plasmid, bacmid, phagemid, cosmid, phage, virus or artificial chromosome.

  Currently preferred in vivo nucleic acid transfer techniques include transfection with adenovirus, lentivirus, type I herpes simplex virus or adeno-associated virus (AAV), and viral or non-viral constructs such as lipid-based systems. Useful lipids for lipid-mediated introduction of genes are, for example, DOTMA, DOPE and DC-Chol [Tonkinson et al., Cancer Investigation, 14 (1): 54-65 (1996)]. The most preferred construct used in gene therapy is a virus, most preferably an adenovirus, AAV, lentivirus or retrovirus. Viral constructs, such as retroviral constructs, can include at least one transcriptional promoter / enhancer or locus-defining element (s) or other such as alternative splicing, nuclear export of nuclear RNA, or post-translational modification of messenger Other elements that regulate gene expression by these means. Such vector constructs also include a packing signal, a long terminal repeat (LTR) or part thereof, and a plus-strand primer binding site and minus suitable for the virus used, if not already present in the viral construct. Contains a strand primer binding site. In addition, such constructs typically include a signal sequence for secretion of the peptide or antibody from the host cell in which it is present. Preferably, the signal sequence for this purpose is a mammalian signal sequence. Optionally, the construct can also include a signal that causes polyadenylation, as well as one or more restriction sites, and a translation termination sequence. By way of example, such constructs typically include a 5 'LTR, a tRNA binding site, a packing signal, an origin of second strand DNA synthesis, and a 3' LTR or a portion thereof. Other vectors that are non-viral can be used (eg, cationic lipids, polylysine and dendrimers).

  Because of the self-aggregating properties of the peptides of the present invention, it is believed that such peptides can also be used as potent detectors of amyloid fibrils / spots in biological samples. This is particularly important for amyloid-related diseases such as Alzheimer's disease, where only a clear diagnosis can be made after brain tissue has been examined postmortem for characteristic neurofibrillary tangles (NFT) and neuritic aspects.

  Thus, according to yet another aspect of the invention, a method is provided for detecting the presence or absence of amyloid fibrils in a biological sample.

  This method involves incubating a biological sample with a peptide of the invention that can co-aggregate with amyloid fibrils, after which the peptide is detected, thereby the presence of amyloid fibrils in the biological sample or This is done by detecting that it does not exist. Various peptide reagents capable of recognizing conformational assemblies are known in the art, some of which are described in Bursavich (2002), J. MoI. Med. Chem. 45 (3): 541-58; Baltzer, Chem. Rev. 101 (10): 3153-63.

  The biological sample utilized for detection can be any body sample such as blood (such as serum or plasma), saliva, ascites, pleural effusion, urine, biopsy sample, isolated cells, and / or cell membranes. It can be a preparation or the like. Various methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.

  The peptides of the invention are contacted with a biological sample under conditions suitable for aggregate formation (ie, buffer, temperature, incubation time, etc.). For example, suitable conditions are described in Example 2 of the Examples section. Various measures are taken to prevent pre-aggregation of the peptide prior to incubation with the biological sample. For this purpose, freshly prepared peptide stocks are preferably used.

  Protein complexes in a biological sample can be detected by any one of several methods known in the art, such as biochemical and / or optical detection. Schemes can be used.

  In order to facilitate detection of the complex, the peptides of the invention are preferably labeled with a tag or antibody. It will be appreciated that labeling can be performed prior to, simultaneously with, or after aggregate formation depending on the labeling method. The term “tag” as used herein refers to a molecule that exhibits a quantifiable activity or property. The tag can be a fluorescent molecule including a chemical fluorophore such as fluorescein or a polypeptide fluorophore (www.clontech.com) such as green fluorescent protein (GFP) or related proteins. In such cases, the tag can be quantified by its fluorescence that is generated upon application of suitable excitation light. Alternatively, the tag can be an epitope tag, ie a very unique polypeptide sequence that allows a specific antibody to bind without substantially cross-reacting with other cellular epitopes. Such epitope tags include Myc tag, Flag tag, His tag, leucine tag, IgG tag, streptavidin tag and the like.

  Alternatively, aggregate detection can be performed with the antibodies of the present invention.

  Thus, this aspect of the invention provides a method for assaying or screening a biological sample, such as a body tissue or fluid suspected of containing amyloid fibrils.

  It will be appreciated that such detection methods can also be utilized in assays to find powerful drugs useful in the prevention or degradation of amyloid deposition. For example, the present invention can be used for high throughput screening of test compounds. Typically, the co-aggregating peptides of the invention are radiolabeled to reduce the assay volume. A competition assay is then performed by monitoring the displacement of the label by the test compound [Han (1996) J. MoI. Am. Chem. Soc. 118: 4506-7; Esler (1996) Chem. 271: 8545-8].

  It will be appreciated that the peptides of the present invention can also be used as potent detectors of amyloid deposition in vivo. Designed peptides that can bind to amyloid deposits are either non-radioactively labeled or labeled with a radioisotope, as described herein above, as is widely known in the art. It can be administered to an individual to diagnose the onset or presence of the discussed amyloid-related disease. Binding after administration of such labeled peptides to amyloid deposits or amyloid-like deposits can be detected by in vivo imaging techniques known in the art.

  The peptides of the present invention can be included in diagnostic or therapeutic kits. For example, a peptide set of a particular disease-related protein or antibody thereto can be packaged in one or more containers with appropriate buffers and preservatives and for diagnostic purposes or to perform therapeutic treatment Can be used for

  Thus, the peptides can each be mixed in a single container, or each can be placed in an individual container. Preferably, the container includes a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container can be formed from a variety of materials such as glass or plastic.

  In addition, other additives such as stabilizers, buffers, blocking agents, etc. can also be added.

  The peptides of such kits can also be bound to solid supports such as beads, array substrates (eg, chips) and used for diagnostic purposes.

  The peptides included in the kit or immobilized on the substrate can be conjugated to a detectable label as described hereinabove.

  The kit also determines whether the subject being tested has a condition, abnormality or disease associated with the amyloid polypeptide of interest, or is at risk of developing such a condition, abnormality or disease. Instructions for clarification can be included.

  Further objects, advantages and novel features of the present invention will be apparent to those skilled in the art from experiments of the following examples, which are not intended to be limiting. Furthermore, each of the various embodiments and aspects described in the invention above and in the claims below are supported by the experiments of the following examples.

  The invention will now be illustrated in a non-limiting manner with reference to the following examples along with the above description.

  In general, the terms used herein and the experimental procedures used in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are explained fully in the literature. See, for example, “Molecular Cloning: A laboratory Manual”, Sambrook et al. (1989); “Current Protocols in Molecular Biology”, Volumes I-III, Ausubel, R., et al. M.M. Eds (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Maryland, (1989); Perbal, “A Practical Guide to ul”. Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al., “Genome Analysis: A Laboratory Manual Series,” Vol. ew York, (1998); methods described in US Pat. Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057; “Cell Biology: A Laboratory Handbook”, I— III, Cellis, J. et al. E. Eds. (1994); “Current Protocols in Immunology”, Volumes I-III, Coligan J. E. Ed., (1994); Stites et al., “Basic and Clinical Immunology”, 8th edition, Appleton & Lange, Norwalk, CT, (1994); Mischel and Shigig Ed, “Selected Methods in Cell.” H. Freeman and Co. New York, (1980); available immunoassays are extensively described in patents and chemical articles, for example, US Pat. Nos. 3,791,932, 3,839,153, 3,850,752, 3850578, 3835987, 3867517, 3879262, 3901654, 3935074, 3984533, 3996345, 4034074, 4098876, 4879219, 5011771, and 5281521; “Oligonucleotide Synthesis”, Gait, M .; J. et al. Ed. (1984); “Nucleic Acid Hybridization”, Hames, B .; D. and Higgins S. J. et al. Ed. (1985); “Transcribation and Translation”, Hames, B .; D. and Higgins S. J. et al. Ed. (1984); “Animal Cell Culture”, Freshney, R .; I. Ed. (1986); “Immobilized Cells and Enzymes”, IRL Press, (1986); “A Practical Guide to Molecular Cloning”, Perbal, B .; , (1984) and “Methods in Enzymology”, Vols. 1-317, Academic Press, “PCR Protocols: A Guide To Methods And Applications, Academic Press, San Di, 19 rd, Academic Press. See Protein Purification and Characterization—A Laboratory Course Manual ”, CSHL Press, (1996), which is incorporated by reference as if fully set forth herein. Other general references are set forth herein. The procedures in the references are considered well known in the art and are described for the convenience of the reader. All the information contained in the references is incorporated herein by reference.

Example 1
Alanine scan of hIAPP basic amyloidogenic unit-principle and peptide synthesis Pancreatic amyloid is found in more than 95% of patients with type II diabetes. Pancreatic amyloid is formed by aggregation of a 37 amino acid long islet amyloid polypeptide (IAPP, GenBank Accession No. gi: 4557655), whose cytotoxicity is directly related to disease development. The formation of IAPP amyloid follows a nucleation-dependent polymerization process that proceeds via a conformational transition from soluble IAPP to aggregated β-sheets. Recently, it has been shown that IAPP hexapeptide (22-27) (NFGAIL, SEQ ID NO: 111) (also called “basic amyloid-forming unit”) is sufficient for the formation of β-sheet-containing amyloid fibrils. [Konstantinos et al. (2000), J. Am. Mol. Biol. 295: 1055-1071].

  To gain further insight into the specific role of the residues that make up this “basic amyloidogenic unit”, a systematic alanine scan was performed. Amino acids were substituted with alanine in order to specifically change the molecular boundaries of the peptide without significantly changing its tendency to form hydrophobic or β-sheet structures. An alanine scan was performed in an area scene specific to human IAPP (Figure 3a). This region contains two serine residues after the NFGAIL motif in the full-length polypeptide. These eight amino acid peptide sequences were used. This is because peptides shorter than this are hydrophobic and therefore have low solubility. FIG. 3b shows a schematic diagram of the chemical structure of the wild-type peptide, while FIG. 3c shows amino acid substitutions in the various mutant peptides produced.

Methods and Reagents—Peptide synthesis was performed by PeptidoGenetic Research & Co. Inc. (Livermore, CA, USA). The peptide sequence was confirmed by ion spray mass spectrometry using a Perkin Elmer Sciex API I spectrometer. The purity of the peptide was determined by reverse phase high pressure liquid chromatography (RP-HPLC) on a _C18 column using a linear gradient of acetonitrile from 10% to 70% in water and 0.1% trifluoroacetic acid (TFA). Confirmed by.

(Example 2)
Aggregation Kinetics of IAPP Peptide Fragments and Variant Derivatives as Monitored by Turbidity Measurement Using Aggregation and Insolubilization Kinetics, Turbidity Measurement at 405 nm to Study Self-Assembly of IAPP Peptide Derived Fragments And monitored.

  Kinetic aggregation assay-A new peptide stock solution was prepared by dissolving lyophilized form of peptide in DMSO (deaggregating solvent) at a concentration of 100 mM. To avoid any prior aggregation, a fresh stock solution was prepared each time before the experiment. The peptide stock solution was diluted in assay buffer and placed in a 96 well plate as follows: 2 μl of peptide stock solution was added to 98 μl of 10 mM Tris (pH 7.2) and in the presence of 2% DMSO. A final concentration of 2 mM of peptide was made. Turbidity data was measured at 405 nm. A buffer solution containing 2% DMSO was used as a blank. Turbidity was measured at room temperature for several time points.

  Results—As shown in FIG. 4a, the wild-type peptide fragment (SEQ ID NO: 1) showed an aggregation kinetic profile very similar to that previously reported for unaged hIAPP hexapeptide [Tenidis et al. ( 2000), J.H. Mol. Biol. 295: 1055-71]. Such profiles strongly indicate a nucleation-dependent polymerization mechanism [Jarrett and Lanbury (1992), Biochemistry, 31: 6865-70]. After a lag time of 20 minutes, the wild type peptide self-assembled into insoluble fibrils. Peptide G3A (SEQ ID NO: 4) showed essentially the same profile as that of the wild-type peptide. The N1A peptide (SEQ ID NO: 2) mediated greater aggregation kinetics despite having a different kinetic profile when compared to the wild-type peptide profile. Interestingly, the aggregation of N1A appeared to be less dependent on nucleation. Substitution of isoleucine or leucine with alanine (peptide I5A (SEQ ID NO: 5) and peptide L6A (SEQ ID NO: 6), respectively) reduced aggregation kinetics but did not completely stop aggregation kinetics. Replacement of the phenylalanine residue with alanine (peptide F2A, SEQ ID NO: 3) resulted in a complete loss of peptide aggregation ability. The F2A peptide was completely soluble in the aqueous assay buffer.

  In summary, kinetic aggregation studies of amyloidogenic fragments suggested a major role for phenylalanine residues in the amyloid formation process by active fragments of IAPP.

(Example 3)
Measuring the average particle size of the aggregates While the turbidity assay also provided important assessments regarding the aggregation ability and kinetics of various peptides, it did not provide information on the size of the actual aggregates formed. The apparent hydrodynamic diameter of the amyloid structure varies due to the irregularity of the amyloid structure, but can nevertheless provide a clear indication as to the extent of the size of the structure formed, A quantitative basis can be given to compare structures formed by different peptides.

  Therefore, the average size of aggregates formed by various peptides was measured using dynamic light scattering (DLS) experiments.

  Method-A freshly prepared peptide stock solution with a concentration of 10 mM was diluted in 10 mM Tris buffer (pH 7.2) and filtered through a 0.2 μm filter to a final concentration of 100 μM peptide and 1% DMSO. . Particle size measurements were performed using a laser source ALV-NIBS / HPPS non-invasive backscatter device. Autocorrection data was approximated using ALV-NIBS / HPPS software to obtain an average apparent hydrodynamic diameter.

  Results—The average apparent hydrodynamic diameter of the structures formed by the various peptides is shown in FIG.

  In summary, the apparent hydrodynamic diameter of the structures formed by the various peptides appeared to be consistent with the results obtained by the turbidity assay. As in the turbidity assay, the wild type peptide and G3A peptide formed particles of very similar hydrodynamic diameter. Smaller structures were observed for N1A, I5A and L6A derivative peptides. Thus, consistent with the turbidity assay, DLS experiments clearly show that no large particles were formed by the F2A peptide under the experimental conditions shown.

Example 4
Test of amyloidogenic ability of wild-type peptides and derivatives by Congo red (CR) binding assay Congo red (CR) staining combined with polarized light microscopy was used to test the amyloidogenicity of the peptides of the present invention. Amyloid fibrils in general, especially fibrillar IAPP, bind to CR and show gold / green birefringence under polarized light [Cooper (1974), Lab. Invest. 31: 232-8; Lanbury (1992), Biochemistry, 31: 6865-70].

  Methods and Reagents—Peptide solutions incubated with 10 mM Tris buffer (pH 7) for 4 days were dried on glass microscope slides. Staining was performed by adding 1 mM CR in 10 mM Tris buffer (pH 7.2) followed by incubation for 1 minute. In order to remove excess CR, the glass slide was washed with double distilled water and dried. A saturated CR solution dissolved in 80% ethanol (v / v) was used for peptides with poor aggregation. In such cases, staining was performed without washing. Birefringence was measured using a WILD Makroskop m420 (x70) equipped with a polarization stage.

  Results-Wild type, N1A and G3A peptides bound CR and showed characteristic green / gold birefringence (Figures 6g, 6a and 6e for normal field and under polarized light) For microscopic observation, see FIGS. 6h, 6b and 6f, respectively). The I5A and L6A peptides bound to CR and exhibited rare but characteristic birefringence (Figs. 6i and 6k for normal field, respectively, and Fig. 6j and Fig. 6l for polarized light, respectively. ). Peptide F2A (NAGAIL) did not show CR binding capacity (FIG. 6c for normal field and FIG. 6d for polarized light). The dried buffer solution stained with CR was used as a negative control (see FIGS. 6m and 6n for normal light and polarization, respectively). Interestingly, no significant difference in binding was observed for the negative control and F2A peptide.

  To demonstrate that the F2A peptide cannot form fibrils, peptide solutions incubated for 14 days were used in the binding assay. Some aggregation was observed visually after “laying” the peptide for 2 weeks, but CR staining did not show any amyloid structure (results not shown). Under the same conditions, incubation of the wild type peptide resulted in significant CR birefringence.

(Example 5)
Ultrastructural analysis of fibril forming peptides and variants The potential fibril forming ability of various peptides was evaluated by electron microscopy analysis.

  Method-Peptide solution (2 mM peptide in 10 mM Tris buffer, pH 7.2) was incubated overnight at room temperature. Fibril formation was evaluated using 10 μl samples placed on a 200 mesh copper grid and covered with a carbon stabilized formvar film (SPI Supplements, West Chester, PA). After incubation for 20-30 seconds, excess liquid was removed and the grid was negatively stained with 2% aqueous uranyl acetate. Samples were examined with a JEOL 1200EX electron microscope operating at 80 kV.

  Results—To further characterize the structures formed by the various peptides, an electron microscopic analysis of negative staining was performed. Consistent with previous results, filament structure was observed for all peptides except for F2A (FIG. 7b), which gave rise to amorphous fibrils (FIGS. 7a-7f). The frequency of fibrils formed by the I5A and L6A peptides (FIGS. 7e and 7f, respectively) is the frequency of the wild-type peptide (FIG. 7d), the N1A peptide and the G3A peptide (FIGS. 7a and 7c, respectively). It was low compared with. Although the EM fields shown for the F2A, I5A and L6A peptides were rarely observed, the results shown by these images confirm the quantitative results shown in the previous section, and thus the fibril morphology Provides qualitative analysis of science.

  The entangled network observed for wild-type, N1A and G3A peptides can be explained by the rapid formation kinetics of these fibrils (see Example 2). A clearer structure and longer fibrils were observed for the I5A and L6A peptides despite the low frequency. These longer fibrils may be the result of slower kinetics that allow for a more ordered fibril organization.

  In summary, the qualitative results of electron microscopy and CR analysis strongly suggest that the phenylalanine residue in the hexaamyloid peptide is very important for its ability to form amyloid.

(Example 6)
Mapping of recognition domains in the basic amyloidogenic unit of hIAPP—principle and MBP-IAPP fusion protein synthesis 28 overlapping overlapping peptides of hIAPP in order to systematically map and compare potential recognition domains (I.e. hIAPP _1-10 , hIAPP _2-11 ,..., HIAPP _28-37 ) were examined for the ability of hIAPP (GenBank accession number gi: 45557665) [Mazor (2002), J . Mol. Biol. 322: 1013-24].

Materials and Experimental Procedures Bacterial strain-E. coli strain TG-1 (Amersham Pharmacia, Sweden) was used for molecular cloning and plasmid propagation. Bacterial strain BL21 (DE3) (Novagen, USA) was used for protein overexpression.

Manipulation of synthetic IAPP and MBP-IAPP fusion proteins-synthetic DNA sequence of human IAPP (SEQ ID NO: 58) modified to include bacterial codon usage, eight overlapping primers (SEQ ID NOs: 50-57) It was prepared by annealing. PCR was performed by 30 cycles of 95 ° C. for 1 minute, 55 ° C. for 1 minute and 72 ° C. for 1 minute. The annealing product was ligated and amplified using the IAPP1 primer (SEQ ID NO: 50) and the IAPP8 primer (SEQ ID NO: 57). Subsequently, an MBP-IAPP (MBP GenBank Accession No. gi: 26554021) fusion sequence was constructed using the IAPP synthesis template, after which this template was constructed using the YAR2 (SEQ ID NO: 60) and YAR1 primers (SEQ ID NO: 59). Was used to insert a V8 Ek cleavage site and a (His) ₆ tag at the N-terminus of IAPP. The two primers contained NotI and NcoI cloning sites, respectively. The resulting PCR product was digested with NcoI and NotI and ligated into the pMALc2x-NN expression vector. The pMALc2x-NN expression vector was constructed by cloning the polylinker site of pMALc-NN ¹⁹ into pMALc2x (New England Biolabs, USA) [BACH (2001), J. Biol. Mol. Biol. 312: 79-93].

Protein expression and purification-E. coli BL21 cells transformed with the expression plasmid pMALc2x-IAPP encoding MBP-IAPP downstream of the strong Ptac promoter were supplemented with 100 μg / ml ampicillin and 1% (W / V) glucose. It was grown on 200 ml of LB medium. Upon reaching an optical density of A ₆₀₀ = 0.8, protein expression was induced with 0.1 mM or 0.5 mM IPTG for 3 hours at 30 ° C.

  Cell extracts were obtained as previously described [Gazit (1999), J. MoI. Biol. Chem. 274: 2652-2657] using freeze-thaw followed by brief sonication, 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 200 mM NaCl, and protease inhibition. Prepared in an agent cocktail (Sigma). The protein extract was clarified by centrifugation at 20,000 g and stored at 4 ° C. MBP-IAPP fusion protein was purified by passing the extract through an amylose resin column (New England Biolabs, USA) and recovered by elution with 20 mM maltose in the same buffer. Purified MBP-IAPP was stored at 4 ° C. Protein concentration was measured using Pierce Coomassie Plus reagent (Pierce, USA) with BSA as a standard. The MBP and MBP-IAPP protein fractions were analyzed on SDS / 12% polyacrylamide gels and the gels were stained with GelCode Blue (Pierce, USA).

  In order to investigate whether the disulfide bond in MBP-IAPP was oxidized, purified MBP protein and MBP-IAPP protein were mixed with 5 equivalents of N-iodoacetyl-N ′-(8-sulfo-1-naphthyl) ethylenediamine ( IAEDANS) (Sigma, Rehovot, Israel) and reacted overnight in the dark at room temperature. The liberated dye was separated from the labeled protein by gel filtration chromatography on a QuickSpin G-25 Sephadex column. Thereafter, the fluorescence of MBP and MBP-IAPP was measured. Only small fluorescent labeling was detected (on average, less than 0.1 probe molecules per protein molecule). There was no significant difference between the labeling of MBP and MBP-IAPP. This suggested that the disulfide bridges in the expressed IAPP molecule were mostly oxidized.

Results Expression and purification of recombinant MBP-IAPP—Before previous attempts to express intact hIAPP in bacteria were successful, the protein was expressed as an MBP fusion that protects hIAPP from undesired aggregation during expression [ Bach (2001), J.A. Mol. Biol. 312: 79-93]. Fusion protein synthesis was performed using bacterial codon usage, as shown in FIG. 8a. The resulting fusion sequence was cloned into pMALc2x-NN as shown in FIG. 8b and introduced into E. coli BL21 (DE3). Growth conditions, cell extract preparation and protein purification were performed as described herein above. Induction with IPTG resulted in high levels of MBP-IAPP accumulation in the soluble fraction, and less than 5% MBP-IAPP fusion protein was found in the insoluble fraction of cell extracts (data not shown) . A portion obtained from a typical purification step for MBP and MBP-IAPP is shown in FIG. As shown, 48 kDa MBP-IAPP had accumulated to 25% of total soluble protein as calculated by scanning a SDS / polyacrylamide gel stained with GelCode Blue with a densitometer. When induced at 30 ° C. in shake flasks (A ₆₀₀ = 2.0), MBP-IAPP accumulated as soluble protein in the cytoplasm at a level of about 150 mg per 1 L of cell culture. Despite the loss during purification, MBP-IAPP was almost uniformly purified with a yield of 80 mg / l · cell. In addition to the factor Xa cleavage site to remove the MBP tag, additional His-Tag was also included for further application of IAPP and convenient homogenization purification (FIG. 8b). This His-Tag can be removed by Ek V8 cleavage at the N-terminal Lys residue of the IAPP sequence, resulting in the release of wild-type IAPP.

(Example 7)
Use hIAPP polypeptides corresponding to successive overlapping sequences identified IAPP peptide array construction _{-HIAPP 1-37} molecular recognition sequence in decamer (SEQ ID NO: 61-88), SPOT technology (Jerini AG, Berlin, Germany) And synthesized on a cellulose membrane matrix. The peptide was covalently linked to a Whatman 50 cellulose support (Whatman, Maidstone, UK) via the C-terminal amino acid. N-terminal acetylation was used for peptide scans because of greater stability against peptide degradation and better presentation of native recognition motifs.

Peptide synthesis—Peptide synthesis was performed as described in Peptron, Inc. (Taejeon, Korea). The peptide sequence was confirmed by ion spray mass spectrometry using the HP1100 series LC / MSD [Hewlett-Packard Company, Palo Alto, CA]. The purity of the peptide was determined using a 30 minute linear gradient of 0% to 100% acetonitrile in water and 0.1% trifluoroacetic acid (TFA) at a flow rate of 1 ml / min using reverse phase on a _C18 column. Confirmed by high pressure liquid chromatography (RP-HPLC).

Binding studies—Peptide arrays on cellulose were first blocked with 5% (V / V) skim milk in Tris-buffered saline (TBS, 20 mM Tris pH 7.5, 150 mM NaCl). The cellulose membrane was then incubated in the same blocking buffer at 4 ° C. for 12 hours in the presence of 10 μg / ml MBP-IAPP _1-37 . The cellulose membrane was then repeatedly washed with 0.05% Tween 20 in TBS. MBP-IAPP _1-37 bound to the cellulose membrane was detected with an anti-MBP monoclonal antibody (Sigma, Israel). HRP-conjugated goat anti-mouse antibody (Jackson Laboratories, USA) was used as the secondary antibody. Immunoblots were developed using Renaisence Western Blot Chemiluminescent Reagent (NEN, USA) according to manufacturer's instructions and signal was quantified using densitometry. Regeneration of the cellulose membrane for reuse is carried out using 62.5 mM Tris, 2% SDS, 100 Mm 2-mercaptoethanol in regeneration buffer I (pH 6.7), 8M urea, 1% SDS, This was performed by sequential washing with regeneration buffer II containing 0.1% 2-mercaptoethanol. The efficiency of the washing step was monitored by contacting the membrane with a chemiluminescent reagent as described.

Results Identification of binding sequences in IAPP polypeptides—corresponding to amino acids 1-10 to amino acids 28-37 of hIAPP _1-37 molecules to identify structural motifs in IAPP molecules that mediate intermolecular recognition between _hIAPP molecules Twenty-eight possible overlapping decamers were synthesized on a cellulose membrane matrix. Peptides conjugated to cellulose membrane were incubated overnight with MBP-hIAPP _1-37 . After washing the cellulose membrane with high salt buffer, immunoblots on the cellulose membrane were analyzed and binding was quantified by densitometry (FIG. 10b). It is understood that the measured binding is semi-quantitative because peptide coupling efficiency during synthesis can vary.

As shown in FIGS. 10a-10b, a number of peptide segments showed binding to MBP-IAPP. Amino acid sequence positioned at the center of the IAPP polypeptide _(i.e., hIAPP _{7~16 ~hIAPP 12~21)} showed the most significant binding to _{MBP-hIAPP 1-37.} It was identified in another binding region of IAPP C-terminal portion _{(hIAPP 19~28 ~hIAPP 21~30),} but binding in this case was less noticeable. In addition, a third binding spot was present in the N-terminal part of IAPP (hIAPP _2-11 ), but in this case the typical distribution near the central motif was not clear. This suggests that this result may be false. Even after over-exposure of the blot (data not shown), no binding in the vicinity of this peptide was detected (for either hIAPP _1-10 or hIAPP _3-12 ). Furthermore, it is believed that the process of fibrillation under physiological conditions is not possible very close to the region 2-11 relative to the disulfide bridge.

  To exclude the involvement of MBP itself in binding to the arrayed peptide, the peptide-bound cellulose membrane was incubated with MBP alone and analyzed by immunoblotting. Binding was not confirmed after membrane development (not shown).

  These results indicate that hIAPP previously revealed binding motif [i.e. basic amyloidogenic unit, hIAPP 20-29, Westermark (1990), Proc. Natl. Acad. Sci. 13: 5036-40; Tenidis (2000), J. MoI. Mol. Biol. 295: 1055-1071; Azriel and Gazit (2001), J. MoI. Biol. Chem. 276: 34156-34161], the major central domain of molecular recognition within hIAPP was identified. The peptide array binding distribution profile (FIG. 10b) suggests that NFVLH (SEQ ID NO: 17) may serve as a core recognition motif.

(Example 8)
Characterization of aggregation kinetics of hIAPP peptide fragments as monitored by turbidimetry. Binding analysis of recombinant MBP-hIAPP fusion protein to hIAPP peptide array (Example 7) shows a putative self-association domain in the central part of hIAPP protein. Identified.

  To identify the smallest structural motif capable of forming amyloid fibrils, a series of peptides contained in this putative self-association domain were tested for aggregation when monitored using turbidity measurements at 405 nm. did.

Table 3 below shows the peptides examined.

Materials and Experimental Procedures Kinetic aggregation assay-A freshly prepared peptide stock solution was made by dissolving lyophilized form of peptide in dimethyl sulfoxide (DMSO) at a concentration of 100 mg / ml. A fresh stock solution was prepared for each experiment to avoid any prior aggregation. The peptide stock solution was diluted in assay buffer as described below in the wells of an enzyme linked immunosorbent assay (ELISA) plate. 8 μl of peptide stock solution was added to 92 μL of 10 mM Tris pH 7.2 (thus the final concentration of peptide was 8 mg / ml in the presence of 8% DMSO). Turbidity data was collected at 405 nm. A buffer solution containing the same amount of DMSO as the test sample was used as a blank and the blank was subtracted from the results. Turbidity was measured continuously at room temperature using a THERMOmax ELISA plate reader (Molecular Devices, Sunnyvale, CA).

Results Turbidity assays were performed to determine the ability of various peptides (Table 3) to aggregate in aqueous media. Fresh stock solutions of various peptide fragments were made in DMSO and then diluted in Tris buffer solution and turbidity was monitored over 2 hours as a measure of protein aggregation. As shown in FIG. 11, NFLVHSS, FLVHSS, and FLVHS peptides showed high turbidity. Amyloid formation by short peptides of NFGAIL has been previously reported in [Tenidis (2000), supra], but the lag time is very short or not at all, and therefore cannot be detected under these experimental conditions, however. It is understood that the aggregation kinetic profile is similar to the aggregation kinetic profile observed for the hexapeptide hIAPP _22-27 (NFGAIL). On the other hand, the peptide NFLVHSSNN shows very slow turbidity, while NFLVH and FLVH show almost no turbidity. Even after a significantly longer incubation, no significant turbidity was observed for the latter two peptides. The absence of amyloid fibril formation is thought to be due to electrostatic repulsion of partially charged histidine residues.

Example 9
Testing amyloidogenicity of hIAPP peptide by Congo red (CP) binding assay Congo red (CP) staining combined with polarizing microscopy was utilized to test the amyloidogenicity of the peptides of the present invention. Amyloid fibrils bind to CR and exhibit gold / green birefringence under polarized light [Puchtler (1966), J. Mol. Histochem. Cytochem. 10: 355-364].

Materials and Experimental Procedures Congo red staining and birefringence—10 μL suspension of 8 mg / ml peptide solution in 10 mM Tris buffer (pH 7.2) aged at least 1 day on a glass microscope slide. Dried overnight. Staining was performed by adding a 10 μL suspension of saturated Congo red (CR) and NaCl in 80% ethanol (v / v) solution as previously described [Puchtler (1966), supra]. The solution was filtered through a 0.45 μm filter. Thereafter, the slide glass was dried for several hours. Birefringence was measured using an SZX-12 stereo microscope (Olympus, Hamburg, Germany) equipped with orthogonal polarizers.

Results A CR birefringence assay was performed to reveal any possible amyloid character of the aggregates formed in the Congo Red staining and birefringence-turbidity assay (see Example 8). Peptide fragments were examined for amyloidogenicity by staining with CR and testing under a light microscope equipped with orthogonal polarizers. Consistent with the kinetic assay results and as shown in FIGS. 12b-c and 12e, the NFLVHSS, FLVHSS and FLVHS peptides showed typical birefringence. In contrast, NFLVHSSNN, NFLVH and FLVH peptides showed very weak birefringence or no birefringence (FIGS. 12a, 12d and 12f). The peptide NFLVHSSNN showed a weaker characteristic birefringence (FIG. 12a). Peptide NFLVH showed strong smeared staining at the edge of the sample (FIG. 12d). Peptide FLVH showed no birefringence (FIG. 12f). To test whether the FLVH peptide did not form amyloid fibrils due to the long lag time, a sample of the peptide solution aged for 5 days was examined. The same peptide was also tested in aqueous solution at a very high concentration (10 mg / ml), but no birefringence was detected in all cases. This indicates that this peptide does not form amyloid (data not shown).

(Example 10)
Ultrastructural analysis of fibril forming hIAPP peptides The potential fibril forming ability of various peptides was evaluated by electron microscopy analysis.

Materials and Experimental Procedures Transmission Electron Microscopy—A 10 μL sample of an 8 mg / ml peptide solution in 10 mM Tris buffer (pH 7.2) aged for at least 1 day was transferred to a 400 mesh copper grid (SPI supplements, West Chester, PA). And covered with a carbon stabilized formvar film. After 1 minute, excess liquid was removed and the grid was then negatively stained with 2% aqueous uranyl acetate for an additional 2 minutes. Samples were examined with a JOEL 1200EX electron microscope operating at 80 kV.

Results Microscopic observation with negative staining was performed to further characterize the structures formed by the various peptides. Consistent with previous results, all peptide fragments showed a fibrillar structure except for FLVH peptides where only amorphous aggregates were found (FIGS. 13a-13f). The NFLVHSSNN peptide showed a long thin coiled filament similar to that formed by the full-length peptide, as described above (FIG. 13a). The peptides NFLVHSS, FLVHSS, FLVHS showed long and wide ribbon-like fibrils as described for the NFGAIL fragment [Tendisis (2000), above; FIGS. 13c-13e, respectively]. The fibrils formed by the NFLVH peptide are thin and short and can be considered protofilaments rather than filaments. Its appearance is much less frequent, and EM pictures represent rather rare events rather than a general field of view (FIG. 13d). As shown in FIG. 13f, the FLVH peptide mediated the formation of amorphous aggregates.

(Example 11)
Secondary structure analysis of hIAPP peptide fragments Fourier transform infrared spectroscopy (FT-IR) was performed to reveal the secondary structure of hIAPP amyloidogenic peptide fibrils and non-fibrillar peptides.

Materials and Experimental Procedures Fourier Transform Infrared Spectroscopy—Infrared spectra were recorded using a Nicolet Nexus 470-IR spectrometer equipped with a DTGS detector. A sample of the aged peptide solution was taken from the turbidity assay and then suspended on a CaF ₂ window (Sigma) plate and vacuum dried. The peptide deposit was resuspended with double distilled water followed by drying to form a thin film. This resuspension procedure was repeated twice to ensure maximum hydrogen to deuterium exchange. Measurements were obtained using 4 cm ⁻¹ resolution and 2000 scan averaging. The transmission minimum was determined by the OMNIC analysis program (Nicolet).

Results FT-IR studies—As shown in FIGS. 14 a-14 f, all fibril peptides have a well-defined minimal band typical for β-sheet structures around 1620 cm ⁻¹ to 1640 cm ^−1. FT-IR spectrum was shown. In contrast, the spectrum of the tetrapeptide FLVH, which has no appearance on fibrils by other methods, is typical of random coil structures. Spectrum of NFLVHSSNN peptides transmittance minimum in the 1621 cm ^-1 to indicate that β- sheet content is large, and showed a minimum at suggesting 1640 cm ^-1 and 1665 cm ^-1 for the presence of non-β structure. Another small minimum was observed at 1688 cm ⁻¹ , indicating an antiparallel β-sheet (FIG. 14a). The spectrum of the NFLVHSS peptide showed large minimal bands at 1929 cm ⁻¹ and 1675 cm ⁻¹ . This spectrum is typical of an antiparallel β-sheet structure (FIG. 14b). Spectrum similar have been observed for the peptide FLVHS with a smaller minimum in a large minimum and 1676cm ^-1 in 1625 cm ^-1 (Fig. 14e). The spectrum of the FLVHSS peptide also showed a large minimum at 1626 cm ⁻¹ . The spectrum also has several small minimum had around ^{1637cm -1 ~1676cm} -1, they had a shape similar to the noise than signal (Figure 14c). The spectrum of the NFLVH peptide showed a minimum at 1636 cm ⁻¹ (which also indicates a β-sheet), but this band, which may indicate the presence of a non-β structure when compared to the other spectra, is 1654 cm ^−1. As well as the observed minimum at 1669 cm ⁻¹ (FIG. 14d). In contrast, the spectrum of FLVH peptide ^shows no minima 1620cm ^-1 ~1640cm -1, showed a number of minimum near the typical ^{1646cm -1 ~1675cm} -1 to random coil structure (Fig. 14f ).

  To examine whether the FLVH tetrapeptide cannot form amyloid fibrils at all, or whether fibril formation that cannot be detected is a result of slow kinetics, a solution of the peptide in the same experimental conditions for 2 months Incubated between and examined for the presence of fibrils. However, no evidence of amyloid fibril formation was detected using EM microscopy, CR staining or FT-IR spectroscopy. These results may suggest that the tetrapeptide cannot form fibrils by considering energy. That is, the energy contribution of stacking chains composed of three peptide bonds is less than the entropy loss of oligomerization.

  In summary, the ultrastructural observations are consistent with findings as revealed by the turbidity assay and the Congo Red birefringence assay. At the same time, experimental data identified a novel pentapeptide element within the hIAPP peptide, ie, the FLVHS peptide, that has strong amyloidogenic potential. Interestingly, although the NFLVH peptide found in the same central domain of hIAPP polypeptide was found to be amyloidogenic, its ability to form fibrils was somewhat inferior.

(Example 12)
Identification of a minimal amyloidogenic peptide fragment of medin Background Medin (GenBank accession number gi: 5174557) is a major component of aortic medial amyloid deposits [Haeggqvist (1999), Proc. Natl. Acad. Sci. USA, 96: 8674-8669]. In previous studies, aortic medial amyloid was found in 97% of patients over the age of 50 [Mucchiano (1992), Am. J. et al. Pathol. 140: 811-877]. However, the pathological role of such amyloid deposits remains unclear. These amyloids have been suggested to play a role in the decreased elasticity of aortic blood vessels associated with aging [Mucchiano (1992), supra; Haeggqvist (1999), supra]. Studies clearly identified the tryptic peptide NGFSVQFV as a medial amyloidogenic peptide, but did not reveal the minimal sequence of peptides that are still amyloidogenic and the molecular determinants that mediate the amyloidogenic process. Such information is important to truly understand the fibrillation process in the specific case of medins, but is also generally important as a paradigm for the amyloid fibril process.

  The smallest active fragment of medins was determined using functional and structural analysis of shortened analogs derived from the published octapeptide [Haegqqvist (1999), supra].

Materials and Experimental Procedures Peptide synthesis was described in Example 7.

Table 4 below shows the peptides examined.

Results To gain further insight into the structural elements of medins that retain the molecular information necessary to mediate the processes of molecular recognition and self-association, a short peptide fragment of medin and its analogs that form amyloid fibrils in vitro. Investigate ability. FIG. 15a shows a schematic diagram of the chemical structure of the largest peptide fragment examined.

(Example 13)
Aggregation Kinetics of Medin Derived Peptide Fragments Turbidity assays were performed as described in Example 8.

  To obtain initial insights on the aggregating ability of various medin-derived peptides, turbidity assays were performed. Freshly made stock solutions of amyloidogenic octapeptide and its truncated analogs were prepared in DMSO. The peptide was then diluted in aqueous solution and turbidity was monitored by following the absorbance at 405 nm as a function of time. As shown in FIG. 16a, NFGSV pentapeptide showed the greatest degree of aggregation within minutes of incubation. Physical testing of the solution showed that the peptide formed a gel structure. The kinetics of the aggregation of NFGSVQV octapeptide was too fast to be measured. This is because turbidity was already observed immediately upon dilution into an aqueous solution (FIGS. 16a-16b). Similar fast kinetics were also observed for the GSVQ tetrapeptide. The shortened peptides of NFGSVQ, FGSVQ and FGSV showed a moderate increase in turbidity over about 30 minutes (FIG. 16b) and then decreased slightly. This can be explained by the sedimentation of large aggregates. In summary, such kinetic and turbidity values were similar to those previously observed for similarly sized amyloidogenic peptides (Azriel and Gazit, 2001).

(Example 14)
Ultrastructural analysis of medin-derived peptide fragments Electron microscopic observation analysis was performed as described in Example 10.

  The fibrillization ability of the medin-derived peptide fragment was performed by electron microscopy (EM) using negative staining. The peptide fragment stock solution was suspended and aged for 4 days. Fibrillar structure was clearly observed in both the solution containing NFGSVQFA octapeptide (FIG. 17a) and the solution containing shortened NFGSVQ (FIG. 17b). In both cases, the structure was similar to that observed for much longer polypeptides, such as IAPP and β-amyloid (Aβ) polypeptides. The shorter gel-forming NFGSV pentapeptide did not form a typical amyloid structure, but formed a fibrous network (FIG. 17c). It should be noted that a fibrous network was recently observed during gelation of glutathione peptides [Lyon and Atkins (2001), J. Mol. Am. Chem. Soc. 123: 4408-4413]. Typical fibrils could not be detected in solutions containing FGSVQ pentapeptide, GSVQ tetrapeptide or FGSV tetrapeptide despite extensive investigation. In the case of the FGSVQ peptide (FIG. 17d), some fibrillar ordered structure can be seen, while significantly different from the structure formed by typical amyloidogenic peptides, but the GSVQ peptide In the case of and FGSV peptides, no fibril structure could be found (FIGS. 17e and 17f, respectively).

(Example 15)
Test of amyloid-forming ability of medyne derived peptides by Congo Red (CR) binding assay. CR staining was performed as described in Example 9.

  CR staining was performed to determine whether the structures formed by various medin-derived peptides exhibited typical birefringence. As shown in FIG. 18b, the NFGSVQ hexapeptide bound to CR and showed a characteristic bright strong green-gold birefringence. The NFGSVQFV octapeptide also showed significant birefringence, although less typical than the birefringence observed for the hexapeptide (FIG. 18a). The gel-forming NFGSV peptide precipitate showed a very low degree of birefringence (FIG. 18c). FGSVQ and FGSV peptides did not show birefringence when stained with CR (FIGS. 18d and 18f, respectively). There was no obvious difference between these two peptides and the negative control (ie, buffer solution without peptide). Interestingly, an unexpectedly high level of birefringence is observed for the GSVQ tetrapeptide (FIG. 18e), while the morphology of the structure formed therefrom (FIG. 18e) is clearly different from that of amyloid fibrils. It was. This indicates that these structures can have a significant degree of order reflected in strong birefringence.

(Example 16)
Effect of Phenylalanine Substitution on Medin Self-Assembly To elucidate the possible role of phenylalanine residues in the process of amyloid fibril formation by minimal amyloidogenic hexapeptides, the amino acid of phenylalanine was substituted with alanine. Alanine substituted peptides were prepared and examined in the same manner as described for the various fragments of medins. As shown in FIG. 19a, markedly reduced turbidity was observed for the alanine substituted peptide when compared to the wild type hexapeptide. Even when the laid solution of NASVSVQ peptide was visualized by EM, no clear fibril structure could be detected (FIG. 19b). This is in stark contrast to the vast fibrillar structure observed for the wild type peptide (Figure 17b). Furthermore, the visualized structure does not show any degree of order as observed for the NFGSV and FGSVQ peptides (FIGS. 17c-17d) as described above, but was observed for the FGSV tetrapeptide (FIG. 17e). Such a completely non-fibrillar structure was very similar. Interestingly, some degree of birefringence can still be detected by the alanine substituted peptide (as observed for the GSVQ peptide (FIG. 18e)) (FIG. 19c). These results raise further doubts that CR staining is used as the sole indicator of amyloid formation [Khurana (2001), J. et al. Biol. Chem. 276: 22715-22721].

  Taken together, these results indicate that the shortened fragment of medin capable of forming amyloid fibrils is the hexapeptide NFGSVQ (SEQ ID NO: 21). However, the shorter pentapeptide fragment NFGSV (SEQ ID NO: 22) showed a fibrous network that was not typical for amyloid. The amyloidogenic NFGSVQ hexapeptide is remarkably similar to the smallest amyloidogenic fragment of the islet amyloid polypeptide (IAPP, see Examples 1-5). Taken together, the results are consistent with the hypothesized role of stacking interactions in the self-association process leading to the formation of amyloid fibrils, and consistent with the suggested correlation between amyloid fibrils and β-helix structures. ing.

(Example 17)
Identification of a minimal amyloidogenic peptide fragment of human calcitonin Human calcitonin (hCT, GenBank Accession No. gi: 179880) is a 32-amino acid-long polymorphic protein involved in calcium homeostasis produced by thyroid C cells. A peptide hormone [Austin and Health (1981), N.A. Engl. J. et al. Med. 304: 269-278; Copp (1970), Annu. Rev. Physiol. 32: 61-86; Zaidi (2002), Bone, 30: 655-663]. It has been found that amyloid fibrils composed of hCT are associated with medullary carcinoma of the thyroid [Kedar (1976), Isr. J. et al. Sci. 12: 1137; Berger (1988), Arch. A. Pathol. Anat. Histopathol. 412: 543-551; Arvinte (1993), J. MoI. Biol. Chem. 268: 6415-6422]. Interestingly, synthetic hCT has been found to form amyloid fibrils in vitro having a morphology similar to deposits found in the thyroid [Kedar (1976), supra; Berger (1988), supra; Arvinte (1993), supra; Benvenge (1994), J. MoI. Endocrinol. Invest. 17: 119-122; Bauer (1994), Biochemistry, 33: 12276-12282; Kanaori (1995), Biochemistry, 34: 12138-43; Kamihara (2000), Protein Sci. 9: 867-877]. The in vitro process of amyloid formation is affected by the pH of the medium [23]. Electron microscope observation experiments have revealed that the fibrils formed by hCT have a diameter of about 80 mm and a length of up to several micrometers. Fibrils are often associated with each other, and in vitro amyloid formation is affected by the pH of the medium [Kamihara (2000), supra].

  Calcitonin is used as a drug for various diseases including Paget's disease and osteoporosis. However, the ability of hCT to associate in aqueous solutions at physiological pH and form amyloid fibrils is a major limitation on its efficient use as a drug [Austin (1981), supra; Copp (1970), supra. Zaidi (2002), supra]. Salmon CT [Zaidi (2002), supra] is a clinically used alternative to hCT, but due to its low sequence homology, it produces an immunogenic response in treated patients. Therefore, understanding the mechanism of amyloid formation by hCT and inhibiting this process is very important not only in the context of the mechanism of amyloid formation, but also as a step toward improved therapeutic use of calcitonin.

Circular dichroism (CD) studies have revealed that in water, monomeric hCT has little ordered secondary structure at room temperature [Arvinte (1993), supra]. However, hCT fibril studies using circular dichroism, fluorescence and infrared spectroscopy reveal that fibrillated hCT molecules have both α-helix and β-sheet secondary structural components. [Bauer (1994), supra]. In NMR spectroscopic studies, in solvents that promote various structures, such as TFE / H ₂ O, hCT adopts an amphiphilic α-helix conformation mainly in the residue range 8-22. [Meadows (1991), Biochemistry, 30: 1247-1254; Mota (1991), Biochemistry, 30: 10444-10450]. In DMSO / H ₂ O, a short double-stranded antiparallel β-sheet morphology in the central region is created by residues 16-21 [Mota (1991), Biochemistry, 30: 2364-71].

  Based on this structural data and the proposed role of aromatic residues in the amyloid formation process, the inventors have identified short peptide fragments that are sufficient to mediate calcitonin self-association [Reches (2002). J. et al. Biol. Chem. 277: 35475-80].

Peptides studied-Based on the previously reported sensitivity of amyloid formation to acidic pH [Kanaori (1995), supra], negatively charged amino acids that undergo protonation at low pH are important in the amyloid formation process. It has been suggested that it can play a role. The only negatively charged amino acid in hCT is Asp ¹⁵ (Figure 20a). In addition, a very important role for the Lys ¹⁸ and Phe ¹⁹ residues in the oligomerization state and biological activity of hCT has recently been shown [Kazantzis (269), Eur. J. et al. Biochem. 269: 780-91]. Together with the presence of two phenylalanine residues in the region, structural analysis of amyloidogenic determinants in hCT was directed to amino acids 15-19. FIG. 20b shows a schematic diagram of the chemical structure of the longest peptide, and Table 5 below shows the various peptide fragments used in this study.

(Example 18)
Ultrastructural analysis of calcitonin-derived peptide fragments Electron microscopic observation analysis was performed as described in Example 10.

  The fibrillation ability of calcitonin-derived peptide fragments was performed by electron microscopy (EM) using negative staining. The peptide fragment stock solution was suspended in 0.02 M NaCl, 0.01 M Tris (pH 7.2), aged for 2 days, and negatively stained. Fibrillar structures formed by full-length polypeptides [Arvinte (1993), supra; Benvenge (1994), supra; Bauer (1994), supra; Kanaori (1995), supra; Kamihara (2000), supra] The fiber structure was clearly and frequently observed in the solution containing DFNKF pentapeptide (FIG. 21a). The shorter DFNK tetrapeptide also formed a fibril structure (FIG. 21b). However, the structure formed was less ordered when compared to the fibril structure formed by DFNKF pentapeptide. The amount of fibril structure formed by DFNK was also lower compared to DFNKF pentapeptide. Obvious fibrils could not be detected using a solution containing FNKF tetrapeptide and DFN tripeptide, despite extensive investigation. In the case of the FNKF tetrapeptide, only amorphous aggregates could be found (FIG. 21c). The DFN tripeptide formed a more ordered structure similar to the structure formed by the gel-forming tripeptide [Lyon (2001), supra] (FIG. 21d). To determine whether the FNKF tetrapeptide and DFN tripeptide peptides are not able to form any fibrils, or whether the observations are the result of slow kinetics, a solution of the peptide is prepared under the same experimental conditions. Incubated for a week. Again, no clear fibril structure could be detected (data not shown).

(Example 19)
Test of amyloidogenic ability of calcitonin-derived peptide by Congo red (CR) binding assay. CR staining was performed as described in Example 9.

  CR staining was performed to determine whether structures formed by various hCT-derived peptides exhibited typical birefringence. As shown in FIGS. 22a-22d, all examined peptides showed some birefringence. However, green birefringence was observed for the DFNKF pentapeptide, which was clear and strong (FIG. 22a). The level of birefringence observed for the other peptides was lower but significant because no birefringence could be detected using a control solution containing no peptide. The lower level of birefringence of the DFNK tetrapeptide (Figure 22b) was consistent with a lower degree of fibrillation, as observed using EM (Figure 21b). However, it is understood that the birefringence observed for FNKF tetrapeptides and DFN tripeptides may represent some orderly structure [Lyon (2001) supra].

(Example 20)
Secondary structure of aggregated hCT-derived peptides FT-IR spectroscopy was performed as described in Example 11.

Amyloid deposits are characteristic of fibrils rich in β-pleated sheet structures. FT-IR spectroscopy was used to obtain quantitative information about the secondary structure formed by various peptide fragments. The aged peptide solution was dried on CaF ₂ plates as described in Example 11 to form a thin film. As shown in FIG. 23, DFNKF pentapeptide (in 1639 cm ^-1 and 1669cm ^-1) denotes a double-minimum, and, consistent with β- sheet structure antiparallel, and amyloid formation of islet amyloid polypeptide The FT-IR spectrum of amide I is shown, which is remarkably similar to the spectrum of the sex hexapeptide fragment [Tendisis (2000), supra]. The spectrum of amide I observed for the DFNK tetrapeptide (FIG. 23) was less typical for β-sheet structures. DFNK tetrapeptide, while showing a minimum at 1666 cm ^-1 which may reflect the antiparallel β- sheet, typical minimum around ^{1620cm -1 ~1640cm} -1 that is typically observed with β- sheet structure Did not have. The FNKF tetrapeptide is typical of an unordered structure and showed an FT-IR spectrum similar to that of a short non-amyloidogenic fragment of the islet amyloid polypeptide [Tendisis (2000), supra] (FIG. 23). ). The DFN tripeptide exhibits a double minimum (at 1642 cm ⁻¹ and 1673 cm ⁻¹ ) (FIG. 23) and shows an FT-IR spectrum of amide I, consistent with a mixture of β-sheet and random structures. It was. This further indicates that the structure observed by EM visualization can represent some orderly structure composed primarily of β-sheet structural elements.

(Example 21)
Effect of phenylalanine substitution on the self-association of calcitonin-derived peptides To insight into the possible role for phenylalanine residues in the calcitonin self-association process, the amino acid of phenylalanine was replaced with alanine in the scene of the pentapeptide (SEQ ID NO: 31). Even when the laid solution of DANKF pentapeptide was visualized by EM, no clear fibril structure could be detected (FIG. 24a). The visualized structure showed some order (when compared to the amorphous aggregates found for FNKF tetrapeptide), but no green-gold birefringence could be observed (FIG. 24b). The FT-IR spectrum of the DANKA pentapeptide is similar to the FT-IR spectrum of the FNKF tetrapeptide and other short non-amyloidogenic peptides, which is typical for unordered structures [Tendisis (2000), supra]. It was. In summary, the effect of phenylalanine to alanine substitution is very similar to the effect of such changes in the scene of short amyloidogenic fragments of islet amyloid polypeptide [Azriel (2001), supra].

  In summary, it has been shown that the hCT-derived pentapeptide (SEQ ID NO: 27) can form well-ordered amyloid fibrils. Typical fibrillar structure as seen by electron microscopy visualization (FIG. 21a), very strong green birefringence when stained with CR (FIG. 22a), and typical antiparallel β-sheet structure ( Figure 23a), all of these show that DFNKF pentapeptide is a very potent amyloidogenic factor. Other pentapeptides that can self-associate are indicated herein above. Nevertheless, with respect to the degree of birefringence and morphology by electron microscopy, the hCT fragment is a potent amyloidogenic fragment of the β-amyloid (Aβ) polypeptide, KLVFFAE [Balbach (2000), Biochemistry, 39: 13748-59] and It appears to be the pentapeptide with the greatest amyloid formation ability. It is believed that electrostatic interactions between opposite charges in lysine and aspartate induce the formation of an ordered antiparallel structure. Interestingly, the DFNK polypeptide showed a significantly reduced ability to form amyloid when compared to the DFNKF peptide. Pentapeptide is thought to be the lower limit for strong amyloidogenic factors. This means that the two pentapeptides of IAPP (NFLVH and FLVHS) can form amyloid fibrils, but their shared common part (tetrapeptide FLVH) cannot form such fibrils. This is consistent with recent results that reveal this (see Examples 1-5).

(Example 22)
Identification of amyloidogenic peptides from lactotransferrin Amyloid fibril formation by lactotransferrin (GenBank accession number gi: 24895280) is associated with familial subepithelial corneal amyloid formation [Sacchettini and Kelly (2002), Nat Rev Drug Discov, 1 : 267-75]. Based on the proposed role of aromatic residues in amyloid self-association, the amyloidogenic properties of lactotransferrin-derived peptide LFNQTG (SEQ ID NO: 32) were investigated.

Materials and Experimental Procedures—Described in Example 7 and Example 10.
Results—To characterize the ability of lactotransferrin-derived peptides to form ultrastructures of fibrillar supramolecules, electron microscopy analysis was performed with negative staining. As shown in FIG. 25, filamentous structures were observed for selected peptides under mild conditions. This suggests that lactoferrin LFNQTG is important for polypeptide self-association. These results further demonstrate that the sequence of amyloidogenic peptides can be predicted by the present invention.

(Example 23)
Identification of Amyloidogenic Peptides from Serum Amyloid A Protein Fragments of serum amyloid A protein (GenBank accession number gi: 134167) have been found in the amyloid state of cases of chronic inflammatory amyloidosis [Westermark et al. (1992), Biochem. Biophys. Res. Commun. 182: 27-33]. Based on the proposed role of aromatic residues in amyloid self-association, the amyloidogenic properties of the serum amyloid A protein-derived peptide SFFSFL (SEQ ID NO: 33) were investigated.

Materials and Experimental Procedures—Described in Example 7 and Example 10.
Results—To characterize the ability of the serum amyloid A protein-derived peptide to form the ultrastructure of fibrillar supramolecules, electron microscopy analysis with negative staining was performed. As shown in FIG. 26, filamentous structures were observed for selected peptides under mild conditions. This suggests that the serum amyloid A protein, SFFSFL, is important for polypeptide self-association. These results further demonstrate that the sequence of amyloidogenic peptides can be predicted by the present invention.

(Example 24)
Identification of amyloidogenic peptides from BriL The human BRI gene is located on chromosome 13. The amyloid fibrils of the BriL gene product (GenBank Accession No. gi: 12643343) are associated with neuronal dysfunction and dementia (Vidal et al. (1999) Nature 399, 776-781). Based on the proposed role of aromatic residues in amyloid self-association, the amyloidogenic properties of the BriL-derived peptide FENKF (SEQ ID NO: 34) were investigated.

Materials and Experimental Procedures—Described in Example 7 and Example 10.
Results—To characterize the BriL-derived peptide's ability to form ultrastructures of fibrillar supramolecules, an electron microscopy observation analysis with negative staining was performed. As shown in FIG. 27, filamentous structures were observed for selected peptides under mild conditions. This suggests that BriL's FENKF is important for polypeptide self-association. These results further demonstrate that the sequence of amyloidogenic peptides can be predicted by the present invention.

(Example 25)
Identification of amyloidogenic peptides from gelsolin A fragment of the gelsolin protein (GenBank accession number gi: 4504165) has been found in the amyloid state of a case of Finnish hereditary amyloidosis [Maury and Nurmiaho-Lasila (1992), Biochem. Biophys. Res. Commun. 183: 227-31]. Based on the proposed role of aromatic residues in amyloid self-association, the amyloidogenic properties of the gelsolin-derived peptide SFNNNG (SEQ ID NO: 35) were investigated.

Materials and Experimental Procedures—Described in Example 7 and Example 10.
Results—To characterize the ability of gelsolin-derived peptides to form ultrastructures of fibrillar supramolecules, electron microscopy analysis was performed with negative staining. As shown in FIG. 28, filamentous structures were observed for selected peptides under mild conditions. This suggests that BriL SFNNG is important for polypeptide self-association. These results further demonstrate that the sequence of amyloidogenic peptides can be predicted by the present invention.

(Example 26)
Identification of amyloidogenic peptides from serum amyloid P Amyloid fibril formation by beta amyloid is promoted by interaction with serum amyloid P (GenBank accession number gi: 2148484). Based on the proposed role of aromatic residues in amyloid self-association, the amyloidogenic properties of serum amyloid P-derived peptide LQNFTL (SEQ ID NO: 36) were investigated.

Materials and Experimental Procedures—Described in Example 7 and Example 10.
Results—To characterize the ability of serum amyloid P-derived peptides to form ultrastructures of fibrillar supramolecules, electron microscopy analysis with negative staining was performed. As shown in FIG. 29, filamentous structures were observed for selected peptides under mild conditions. This suggests that LQNFTL of serum amyloid P is important for polypeptide self-association. These results further demonstrate that the sequence of amyloidogenic peptides can be predicted by the present invention.

(Example 27)
Identification of amyloidogenic peptides from immunoglobulin light chains Formation of amyloid fibrils by immunoglobulin light chains (GenBank accession number gi: 625508) is associated with primary systemic amyloidosis [Sacchettini and Kelly (2002), Nat Rev Drug Discov 1: 267-75]. Based on the proposed role of aromatic residues in amyloid self-association, the amyloidogenic properties of the immunoglobulin light chain derived peptide TLIFGG (SEQ ID NO: 37) were investigated.

Materials and Experimental Procedures—Described in Example 7 and Example 10.
Results—To characterize the ability of the immunoglobulin light chain-derived peptide to form the ultrastructure of the fibrillar supramolecule, an electron microscopic analysis with negative staining was performed. As shown in FIG. 30, filamentous structures were observed for selected peptides under mild conditions. This suggests that the immunoglobulin light chain TLIFGG is important for polypeptide self-association. These results further demonstrate that the sequence of amyloidogenic peptides can be predicted by the present invention.

(Example 28)
Identification of amyloidogenic peptides from cystatin C The formation of amyloid fibrils by cystatin C (GenBank accession number gi: 4490944) is associated with hereditary brain amyloid angiopathy [Sacchettini and Kelly (2002), Nat Rev Drug Discov 1: 267-75]. Based on the proposed role of aromatic residues in amyloid self-association, the amyloidogenic properties of the cystatin C-derived peptide RALDFA (SEQ ID NO: 38) were investigated.

Materials and Experimental Procedures—Described in Example 7 and Example 10.
Results—To characterize the ability of the cystatin C-derived peptide to form the ultrastructure of fibrillar supramolecules, an electron microscopic observation analysis with negative staining was performed. As shown in FIG. 31, filamentous structures were observed for selected peptides under mild conditions. This suggests that cystatin C RALDFA is important for polypeptide self-association. These results further demonstrate that the sequence of amyloidogenic peptides can be predicted by the present invention.

(Example 29)
Identification of amyloidogenic peptides from transthyretin The formation of amyloid fibrils by transthyretin (GenBank Accession No. gi: 72095) is associated with familial amyloid polyneuropathy [Sacchettini and Kelly (2002), Nat Rev Drug Discov 1: 267-75]. Based on the proposed role of aromatic residues in amyloid self-association, the amyloidogenic properties of the transthyretin-derived peptide GLVVFVS (SEQ ID NO: 39) were investigated.

Materials and Experimental Procedures—Described in Example 7 and Example 10.
Results—To characterize the ability of the transthyretin-derived peptide to form the ultrastructure of fibrillar supramolecules, electron microscopy analysis with negative staining was performed. As shown in FIG. 32, filamentous structures were observed for selected peptides under mild conditions. This suggests that the transthyretin GLVFVS is important for polypeptide self-association. These results further demonstrate that the sequence of amyloidogenic peptides can be predicted by the present invention.

(Example 30)
Identification of amyloidogenic peptides from lysozyme Formation of amyloid fibrils by lysozyme (GenBank accession number gi: 299033) is associated with familial non-neuropathic amyloidosis [Sacchettini and Kelly (2002), Nat Rev Drug Discov, 1: 267-75]. Based on the proposed role of aromatic residues in amyloid self-association, the amyloidogenic properties of the lysozyme-derived peptide GTFQIN (SEQ ID NO: 40) were investigated.

Materials and Experimental Procedures—Described in Example 7 and Example 10.
Results—To characterize the ability of lysozyme-derived peptides to form ultrastructures of fibrillar supramolecules, electron microscopic observation analysis with negative staining was performed. As shown in FIG. 33, filamentous structures were observed for selected peptides under mild conditions. This suggests that lysozyme GTFQIN is important for polypeptide self-association. These results further demonstrate that the sequence of amyloidogenic peptides can be predicted by the present invention.

(Example 31)
Identification of Amyloidogenic Peptides from Fibrinogen Formation of amyloid fibrils by fibrinogen (GenBank Accession No. gi: 11761629) is associated with hereditary renal amyloidosis [Sacchettini and Kelly (2002), Nat Rev Drug Discov, 1: 267-75. ]. Based on the proposed role of aromatic residues in amyloid self-association, the amyloidogenic properties of the fibrinogen-derived peptide SGIFTN (SEQ ID NO: 41) were investigated.

Materials and Experimental Procedures—Described in Example 7 and Example 10.
Results—To characterize the ability of fibrinogen-derived peptides to form ultrastructures of fibrillar supramolecules, electron microscopic analysis with negative staining was performed. As shown in FIG. 34, filamentous structures were observed for selected peptides under mild conditions. This suggests that the fibrinogen SGIFTN is important for polypeptide self-association. These results further demonstrate that the sequence of amyloidogenic peptides can be predicted by the present invention.

(Example 32)
Identification of amyloidogenic peptides from insulin The formation of amyloid fibrils by insulin (GenBank accession number gi: 229122) is associated with injection-localized amyloidosis [Sacchettini and Kelly (2002), Nat Rev Drug Discov, 1: 267-75. ]. Based on the proposed role of aromatic residues in amyloid self-association, the amyloidogenic properties of the insulin-derived peptide ERGFF (SEQ ID NO: 42) were investigated.

Materials and Experimental Procedures—Described in Example 7 and Example 10.
Results—To characterize the ability of insulin-derived peptides to form ultrastructures of fibrillar supramolecules, electron microscopy analysis with negative staining was performed. As shown in FIG. 35, filamentous structures were observed for selected peptides under mild conditions. This suggests that the insulin ERGFF is important for polypeptide self-association. These results further demonstrate that the sequence of amyloidogenic peptides can be predicted by the present invention.

(Example 33)
Identification of amyloidogenic peptides from prolactin Formation of amyloid fibrils by prolactin (GenBank Accession No. gi: 4505065) is associated with pituitary amyloidosis [Sacchettini and Kelly (2002), Nat Rev Drug Discov, 1: 267-75]. . Based on the proposed role of aromatic residues in amyloid self-association, the amyloidogenic properties of the prolactin-derived peptide RDFLDR (SEQ ID NO: 43) were investigated.

Materials and Experimental Procedures—Described in Example 7 and Example 10.
Results—To characterize the ability of prolactin-derived peptides to form ultrastructures of fibrillar supramolecules, electron microscopy analysis was performed with negative staining. As shown in FIG. 36, filamentous structures were observed for selected peptides under mild conditions. This suggests that the prolactin RDFLDR is important for polypeptide self-association. These results further demonstrate that the sequence of amyloidogenic peptides can be predicted by the present invention.

(Example 34)
Identification of amyloidogenic peptides from beta-2-microtubulin The formation of amyloid fibrils by beta-2-microtubulin (GenBank accession number gi: 70065) is associated with hemodialysis-related amyloidosis [Sacchettini and Kelly ( 2002), Nat Rev Drug Discov, 1: 267-75]. Based on the proposed role of aromatic residues in amyloid self-association, the amyloidogenic properties of the beta-2-microtubulin derived peptide SNFLN (SEQ ID NO: 44) were investigated.

Materials and Experimental Procedures—Described in Example 7 and Example 10.
Results—To characterize the ability of beta-2-microtubulin-derived peptides to form ultrastructures of fibrillar supramolecules, electron microscopy analysis with negative staining was performed. As shown in FIG. 37, filamentous structures were observed for selected peptides under mild conditions. This suggests that the beta-2-microtubulin SNFLN is important for polypeptide self-association. These results further demonstrate that the sequence of amyloidogenic peptides can be predicted by the present invention.

(Example 35)
Inhibition of amyloid formation of amyloidogenic peptides identified according to the teachings of the present invention The ability of the amyloidogenic peptides of IAPP identified according to the teachings of the present invention to inhibit amyloid formation by full-length polypeptides is represented in the peptide sequence NFLVHPP (SEQ ID NO: 45). As shown, the proline residue of the β-breaker was examined by adding it to the recognition sequence.

  The extent of amyloid fibril formation in the presence and absence of inhibitor was assessed using thioflavin T (ThT) as a molecular indicator. The degree of fluorescence of the ThT dye directly correlates with the amount of amyloid fibrils in solution [LeVine H 3rd. (1993), Protein Sci. 2: 404-410]. IAPP solution (4 μM hIAPP in 10 mM Tris buffer (pH 7.2)) was incubated at room temperature in the presence or absence of 40 μM modified peptide (ie, NFLVHPP). This solution was diluted 10-fold into a solution containing 3 μM Thioflavin T (ThT) in 50 mM sodium phosphate (pH 6.0) and used at 450 nm using an LS50B spectrofluorometer (Perkin Elmer, Wellesley, Mass.). Fibril formation was measured by measuring fluorescence at 480 nm upon excitation. As a control, 10 mM Tris buffer (pH 7.2) was diluted in ThT solution and fluorescence was measured as described.

  Results—As shown in FIG. 38, IAPP alone showed a high level of ThT fluorescence as expected for amyloidogenic proteins, but significantly increased fluorescence in the presence of inhibitory peptides. Therefore, these results confirm the NFLVH sequence as a determinant of amyloid formation in the IAPP polypeptide.

(Example 36)
Importance of hydrophobic residues in amyloid association The importance of aromatic residues in the basic amyloid-forming unit of IAPP was demonstrated in Examples 1-5. As noted, substitution of phenylalanine for alanine abolished the ability of amyloidogenic fragments (NAGAIL, SEQ ID NO: 9) to form amyloid fibrils in vitro. As a result of this observation, a prominent presence of aromatic residues in other short amyloid-related sequences (Examples 12-35) and the well-known role of π-stacking in chemistry and biochemistry self-association processes Based on the above, it has been suggested that stacking of aromatic residues may play a role in the amyloid fibril formation process [Gazit (2002), FASEB J. et al. 16: 77-83].

  The study was further extended to show whether phenylalanine residues are important for their hydrophobic nature rather than for their aromaticity. The effect of phenylalanine substitution by hydrophobic residues on the self-association of the basic amyloid-forming unit of IAPP (ie, NFGAIL peptide) was examined.

A list of peptides used in this study and their display is shown in Table 6 below.

  These hydrophobic amino acids have similar hydrophobicity to phenylalanine or are slightly more hydrophobic than phenylalanine [Wolfenden (1981), Biochemistry, 20: 849-855; Kyte (1982), J. Am. Mol. Biol. 157: 105-132; Radzica (1988)], it is understood that they are not aromatic. Furthermore, valine and isoleucine are considered to be very strong β-sheet formers that are presumed to be important for the formation of β-sheet rich amyloid fibrils [Chou (1974), Biochemistry, 13: 211-222; Chou (1978), Annu. Rev. Biochem. 47: 251-276].

(Example 37)
Characterization of aggregation kinetics of hydrophobically modified hIAPP peptide fragments as monitored by turbidity measurements Experimental procedure-performed as described in Example 8.
Results A turbidity assay was performed in order to gain insight into the aggregating ability of hydrophobically modified IAPP-derived peptide analogs. Freshly made stock solutions of wild type peptides and various peptide variants were made in DMSO. The peptide was then diluted in buffer solution and turbidity was monitored by following the absorbance at 405 nm as a function of time. As shown in FIG. 39, a significant increase in turbidity was observed for the wild type NFGAILSS octapeptide within minutes after its dilution in aqueous solution. The shape of the aggregation curve resembled the shape of the saturation curve with a rapid increase in turbidity present in the first hour followed by a much slower increase in turbidity throughout the monitored incubation time. This probably reflects a rapid aggregation process with the number of free components as the rate limiting factor. In contrast, none of the analog peptides revealed any meaningful aggregation behavior, and the turbidity of all hydrophobic analogs as well as alanine substituted analogs remained very low for at least 24 hours (FIG. 39).

  In order to determine whether the non-aggregation behavior of hydrophobic analogs is a result of very slow kinetics, peptide analog solutions were incubated for 1 week under the same experimental conditions and endpoint turbidity values were measured. As shown in FIG. 30, a somewhat lower degree of turbidity was observed for NIGAILSS, and a lower degree was observed for NLGAILSS, NAGAILSS and NVGAILSS peptides in order of decreasing turbidity. However, even in the case of NIGAILSS, the degree of turbidity was significantly reduced compared to the wild type NFGAILSS protein (FIG. 40). In addition, since a lower degree of aggregation was observed for the replacement of the highly hydrophobic β-sheet forming factor with valine, there was no correlation between aggregation ability and hydrophobicity or β-sheet formation tendency. There wasn't. A slight decrease in the end point turbidity value of the NFGAILSS wild-type peptide may reflect the formation of very large aggregates attached to the cuvette surface when compared to the value obtained after 24 hours of incubation.

(Example 38)
Ultrastructural analysis of hIAPP peptide fragments modified to hydrophobicity Electron microscopy analysis was performed as described in Example 10.
Ultrastructural visualization of any possible structure formed by the various analog peptides was performed after 5 days of incubation. This structural analysis represents the most sensitive method because it visualizes the various aggregates individually. To that end, the presence and characteristics of the structures formed were examined by electron microscopy using negative staining with the same peptide solution incubated in the aggregation assay (Example 32). As expected, well-ordered fibrils were observed for the NFGAILSS peptide fragment of the wild-type peptide (FIGS. 41a-41b). Some amorphous aggregates could also be seen for the modified fragments (FIGS. 41c-41f). However, their structure was not significantly greater on the microscope grid. Larger aggregating structures were observed for the more hydrophobic substitutions when compared to the alanine analog. Nevertheless, as noted above, unlike the ordered fibrillar structure observed for the NFGAILSS peptide, these aggregates were extremely rare and did not have an ordered structure (Figures 41c-41). 41f). These irregular and sporadic structures are consistent with some degree of non-specific aggregation as expected after long incubations of fairly hydrophobic molecules.

(Example 39)
Determination of the specific function of phenylalanine in IAPP self-association To determine the specific role of phenylalanine residues in IAPP self-association, a membrane-type binding assay was performed using a full-length hIAPP that recognizes the “basic amyloidogenic unit”. This was done to systematically investigate the molecular determinants that promote the ability. For this purpose, the ability of MBP-IAPP (see Example 6) to interact with an array of peptides (SEQ ID NOs: 91-110) with systematically altered phenylalanine positions was examined.

Materials and Experimental Procedures-See Examples 6-7.
Results A peptide array corresponding to the SNNXGAILSS motif (SEQ ID NO: 90) (where X is any natural amino acid except cysteine) was constructed. As shown in FIG. 42a, MBP-IAPP binding was clearly observed for peptides containing aromatic tryptophan and phenylalanine residues in the X position (FIG. 42a). Interestingly, binding was also observed when phenylalanine was replaced with basic amino acids such as arginine and lysine. In contrast, no binding was observed for any of the hydrophobic substitutions at that position, even after prolonged exposure to the membrane (FIG. 42b).

  Binding with short exposures was assessed using densitometry (Figure 42c). However, it is understood that the measured binding must be interpreted as semi-quantitative because the coupling efficiency during synthesis, and thus the amount of peptide per spot, can vary. However, in this case it was very clear that the difference in binding between the various peptide variants was significant.

  Taken together, all of these observations demonstrate the role of aromatic residues in promoting the amyloid formation process.

(Example 40)
Design and conformation of α-aminoisobutyric acid (Aib) substituted amyloidogenic peptides The minimal amyloidogenic region of the IAPP polypeptide (ie, IAPP14-20, see Table 3 above) binds to and blocks it, It was selected as a target sequence for designing inhibitors that can prevent aggregation.

Experimental approach In order to eliminate the ability of amyloidogenic peptides to aggregate, a β-sheet disrupter can be incorporated into the target sequence and the peptide cannot exhibit a β-sheet conformation in which monomers are stacked together to form fibrils Like that. α-Aminoisobutyric acid (Aib) is an unnatural amino acid containing two methyl residues attached to C _α of the carboxyl group. Unlike natural amino acids, the molecule has no bonded hydrogen atoms in C _alpha. This has a wide influence on the steric properties of amino acids, particularly with respect to the φ and ψ angles of amide bonds. Alanine has a wide range of possible φ and ψ conformations, while Aib, an α-methylated alanine, has a limited φ and ψ conformation. FIG. 43a shows the Aib conformational map derived from the overlap of L-alanine and D-alanine Ramachandran plots. As is clear for FIG. 43a, the possible angles are limited to a small region, and the overall structure is much better for the α-helix conformation rather than the β-strand conformation.

  Thus, Aib can be used to prevent the β-sheet conformation that is the center of amyloid aggregation. In particular, a comparison between the Aib and proline Ramachandran plots shows that Aib is a more potent β-sheet breaker than proline (FIG. 43a).

  Two peptides containing the IAPP amyloid-forming region (ie, ANFLVH and ANFLV, SEQ ID NOs: 124 and 126, respectively) were synthesized to include Aib replacing alanine and leucine residues. Newly synthesized peptides containing the following amino acid sequences, namely Aib-NF-Aib-VH (SEQ ID NO: 125) and Aib-NF-Aib-V (SEQ ID NO: 127), are shown in FIGS. 43b-c, respectively.

Peptide synthesis-Peptron, Inc. using solid phase technology. Peptides were synthesized by (Taejeon, Korea). The exact identity of the peptide was confirmed by ion spray mass spectrometry using the HP1100 series LC / MSD. The purity of the peptide was measured in reverse phase on a _C18 column using a 30 minute linear gradient of acetonitrile from 0% to 100% in water and 0.1% trifluoroacetic acid (TFA) at a flow rate of 1 ml / min. Confirmed by high pressure liquid chromatography (RP-HPLC).

  Peptide solution—A freshly prepared stock solution was made by dissolving a lyophilized form of the peptide in dimethyl sulfoxide (DMSO) at a concentration of 100 mM. A fresh stock solution was prepared for each experiment to avoid any prior aggregation. The peptide stock solution was diluted into microtubes as described below. 5 μl of peptide stock solution was added to 95 μL of 10 mM Tris pH 7.2 (thus the final concentration of peptide was 5 mM in the presence of 5% DMSO).

(Example 41)
Ultrastructural analysis of wild type and Aib modified hIAPP peptides The fibril forming ability of the peptide described in Example 40 above was evaluated by electron microscopy analysis.

Materials and Experimental Procedures Transmission Electron Microscope-A 10 μL sample of 5 mM peptide solution in 10 mM Tris buffer (pH 7.2) aged for 4 days (wt peptide) and 10 days (modified peptide) was transferred to a 400 mesh copper grid (SPI (Supplies, West Chest PA) and covered with a carbon stabilized formvar film. After 1 minute, excess liquid was removed, and the grid was then negatively stained with 2% aqueous uranyl acetate for an additional 2 minutes. Samples were examined with a JEOL 1200EX electron microscope operating at 80 kV.

Results The ability of Aib-containing peptides to form amyloid fibrils was examined in comparison to the native IAPP peptide. The laid solution of Aib modified and wild type peptide was examined under electron microscopy (EM) using negative staining. As shown in FIGS. 44a-b, both natural peptides, ANFLVH (FIG. 44a) and ANFLV (FIG. 44b), formed a fibril structure with high similarity to the fibrils formed by the full-length IAPP protein. . On the other hand, for Aib-containing peptides, ie, Aib-NF-Aib-VH (FIG. 44c) and Aib-NF-Aib-V (FIG. 44d), no fibrillar structure was observed even after a longer incubation period. Although not present, amorphous aggregates were still present. This suggests that the Aib-substituted peptide of the present invention cannot form fibrils.

(Example 42)
Testing amyloidogenic ability of wild-type and Aib-substituted IAPP peptides by Congo red binding assay Materials and experimental procedures Congo red staining and birefringence—10 μL of 5 mM peptide solution in 10 mM Tris buffer (pH 7.2) aged for 10 days The suspension was dried overnight on a glass microscope slide. Staining was performed by adding a 10 μL suspension of saturated Congo Red (CR) and NaCl in 80% ethanol (v / v) solution. The solution was filtered through a 0.2 μm filter. Thereafter, the slide glass was dried for several hours. Birefringence was measured using an SZX-12 stereo microscope (Olympus, Hamburg, Germany) equipped with orthogonal polarizers. Shown at 100 × magnification.

Results Congo red staining was used to evaluate the ability of Aib modified peptides to form amyloid. The slide glass was examined under a microscope using an orthogonal polarizer. Figures 45a-b show typical yellow-green birefringence for both ANFLVH and ANFLV peptides (Figures 45a and b, respectively). However, according to EM studies, the Aib modified peptides, Aib-NF-Aib-VH and Aib-NF-Aib-V, did not show any birefringence. This suggests that the Aib modified peptide cannot form amyloid fibrils at all (FIGS. 45c-d).

(Example 43)
Secondary structure analysis of Aib modified IAPP peptide fragments Fourier transform infrared spectroscopy (FT-IR) was performed to determine the secondary structure of Aib modified hIAPP.

Materials and Experimental Procedures Fourier Transform Infrared Spectroscopy—Infrared spectra were recorded using a Nicolet Nexus 470-IR spectrometer equipped with a DTGS detector. A sample of the peptide solution aged for 2 weeks was suspended on a CaF ₂ plate and vacuum dried. The peptide deposit was resuspended with D ₂ O and subsequently dried to form a thin film. This resuspension procedure was repeated twice to ensure maximum hydrogen to deuterium exchange. Measurements were obtained using 4 cm ⁻¹ resolution and 2000 scan averaging. The transmission minimum was determined by the OMNIC analysis program (Nicolet).

Results FT-IR spectroscopy was used to elucidate the internal conformation of the observed structure (see Examples 42 and 43 above). As shown in FIGS. 46a-b when Aib was contained, the IAPP peptide showed a sharp change in the IR spectrum. The ANFLVH and ANFLV peptide spectra are typical of β-sheet spectra with local minima at 1630 cm ⁻¹ and 1632 cm ⁻¹ , respectively, while Aib-NF-Aib-VH and Aib-NF-Aib-V peptides are 1670 cm ^{− A} minimum is shown for each of ¹ and 1666 cm ⁻¹ , which is characterized by a disordered coil conformation.

  Taken together, these results suggest a fundamental difference between the native IAPP peptide and the Aib-containing peptide. Natural peptides are highly amyloidogenic, but modified Aib-containing peptides are unable to form amyloid fibrils (Examples 41-43).

(Example 44)
Aib-modified peptides inhibit amyloid formation by IAPP polypeptides The extent of amyloid fibril formation in the presence and absence of Aib inhibitors was evaluated using thioflavin T (ThT) as a molecular indicator. The degree of fluorescence of the ThT dye directly correlates with the amount of amyloid fibrils in solution [LeVine H 3rd. (1993), Protein Sci. 2: 404-410].

Materials and Experimental Procedures Fourier Transform Infrared Spectroscopy—IAPP solution (4 μM peptide in 10 mM Tris buffer (pH 7.2)) was incubated at room temperature in the presence or absence of 40 μM various peptide solutions. The solution is diluted 10-fold into a solution containing 3 μM Thioflavin T (ThT) in 50 mM sodium phosphate (pH 6.0) and fluorescence at 480 nm is measured by excitation at 450 nm using a Perkin Elmar LS50B spectrofluorometer Fibril formation was measured. As a control, 10 mM Tris buffer (pH 7.2) was diluted in a ThT solution and fluorescence was measured as described.

Results As shown in FIG. 47, all Aib modified peptides were able to inhibit the association of full-length IAPP polypeptides. This suggests that Aib modification of amyloidogenic peptides can serve as a powerful inhibitory tool in various therapeutic applications.

(Example 45)
Di- and tri-aromatic peptides can inhibit the aggregation of IAPP polypeptides.
To study the ability of short aromatic amino acid sequences to inhibit amyloid polypeptide association and to approach the ability of β-sheet breaker amino acids to facilitate such inhibition, an array of tetra-, tri- and dipeptides is used. Were synthesized and evaluated.

Experimental Procedure Peptide Synthesis-A list of peptides used in this Example 45 and Examples 46-47 and their display is shown in Table 7 below. Peptide synthesis is described in Examples 46-47 below.

ThT fluorescence-see Example 44 above Results Inhibitory peptides were evaluated using a standard ThT fluorescence assay (as described). FIG. 48 shows the endpoint values after 142 hours of incubation after the IAPP aggregation reached a plateau. Aggregation assays were performed in the presence of IAPP polypeptide (4 μM) and inhibitory peptide (40 μM).

  As clearly shown in FIG. 48, the short aromatic amino acid sequence mediates recognition for the IAPP polypeptide while inhibiting its aggregation. The best inhibitor is the Aib-Phe-Phe peptide (EG12), where the Aib residue is bound to the shortest recognition element that can form an amyloid-related structure [Reches and Gazit, Science (2003), 300 (2619): 625-7]. D-Tyr-D-Pro (EG18) and D-Tyr-D-Aib (EG16) show a significant inhibitory effect on IAPP aggregation, which is the ability of a single aromatic residue to mediate molecular recognition To demonstrate. Interestingly, Aib showed higher inhibitory activity than proline. Furthermore, the significant difference in activity between peptides EG17 and EG18 that differ in the position of the β-sheet breaker amino acid relative to the aromatic amino acid suggests a role for order as well as peptide composition.

(Example 46)
Selection of the best performing IAPP fibrillation inhibitor and criteria for selecting it Peptide synthesis-Peptide synthesis (except EG5, EG6 and EG7, D-Phe-Pro, and D-Pro-Phe), solid phase synthesis (Peptron, Inc., Taejeon, Korea). The identity of the peptide was confirmed by ion spray mass spectrometry. The purity of the peptide was confirmed by reverse phase high pressure liquid chromatography (RP-HPLC). EG5, EG6, and EG7, D-Phe-Pro, and D-Pro-Phe were purchased from Bachem (Bubendorf, Switzerland). Islet amyloid polypeptide (IAPP) was purchased from CalBiochem (La Jolla CA, USA).

Thioflavin fluorescence assay-hIAPP fibrillation was monitored by a thioflavin T dye binding assay. The hIAPP _1-37 stock solution was diluted to a final concentration of 4 μM and a final concentration of 1% (vol) HFIP in 10 mM sodium acetate buffer (pH 6.5) with or without inhibitor (40 μM). Immediately after dilution, the sample was centrifuged at 20000 × g for 20 minutes at 4 ° C. and the supernatant fraction was used for fluorescence measurements. For each measurement, Tht was added to a 1 ml sample at a final concentration of 3 μM and the measurement was performed using a Perkin-Elmer (excitation 450 nm, 2.5 nm slit; emission 480 nm, 10 nm slit). Background radiation was subtracted from all samples.

Results Rules for Identification of Potential Inhibitors and Inhibitor Design-Peptide Selection to Identify Small Inhibitors of IAPP Fibrilization and Optimize Inhibitor Minimum Length and Its Maximum Stability Repeated cycles of were performed using D-amino acid sequence analogs.

  The first round of selection shown in FIG. 49a demonstrated that short peptides, such as tripeptides, can effectively inhibit amyloid formation by IAPP. Comparison of EG01 and EG03 suggests that the presence of Asn residues in the short peptide does not contribute to inhibition of amyloid formation and further supports the use of aromatic moieties with β-breakers for proper inhibition To do. EG05, a known inhibitor for amyloid formation [Tjerenberg et al. (1996), J. MoI. Biol. Chem. 271: 8545-854], an effective inhibition of IAPP fibrillation suggests a general inhibition of amyloid formation by aromatic residues. Indeed, similar activity of EG05 and the much shorter EG08 (Aib bound to Phe-Phe) element clearly suggests that the general inhibition of EG05 is due to its aromatic nature.

  The second round of selection shown in FIG. 49b demonstrated that effective inhibition can be achieved not only by tripeptides but also by dipeptides (EG16, EG17, EG18). In all cases, the coupling of the D-isomer to the aromatic moiety was used. The difference between EG16 and EG17 was further studied in the next round to establish rules for inhibitor design. FIG. 49c shows the result of the third round of selection. A comparison in the third round of EG20 vs. EG21 (dFP vs. Pdf) (FIG. 49c) shows the results obtained by comparing EG16 and EG17 in the second round, and EG24 and EG35. Together with the results obtained in the comparison (in the fourth round-Fig. 1d), suggest a general formula for the design of dipeptide inhibitors as described in (Aromatic D or L)-(β-destroying agents) To do. These selection steps further provided a general formula for tripeptide inhibitors as described in (Aromatic D or L)-(Aromatic D or L)-(β-Disruptor).

  The fourth round also demonstrated the ability to inhibit the four putative metabolically stable dipeptides EG28, EG29, EG30, EG31. Again, the usefulness of aromatic amino acids and beta-breaking agents for inhibitory sequences is emphasized.

(Example 47)
Inhibition of β-amyloid polypeptide fibril formation by D-Trp-Aib Peptide synthesis—See Example 46 above. Recombinant β-amyloid (Aβ1-40,> 98% purity) was purchased from rPeptide (Athens GA, USA).

  Thioflavin T fluorescence assay—Aβ1-40 fibrillation was monitored by a thioflavin T dye binding assay. Aβ1-40 stock solution was diluted to a final concentration of 5 μM in 100 mM NaCl, 10 mM sodium phosphate buffer (pH 7.4) with or without inhibitors. For each measurement, Tht was added to a 0.1 ml sample to a final concentration of 0.3 μM ThT and 0.4 μM polypeptide. Measurements were performed using Jobin Yvon FluoroMax-3 (excitation 450 nm, 2.5 nm slit; emission 480 nm, 5 nm slit, integration time 1 second). Background radiation was subtracted from all samples.

  Transmission Electron Microscopy—A 10 μL sample was placed on a 400 mesh copper grid (SPI supplies, West Chester PA) and covered with a carbon stabilized formvar film. After 1 minute, excess liquid was removed and the grid was then negatively stained with 2% aqueous uranyl acetate for an additional 2 minutes. Samples were examined with a JOEL 1200EX electron microscope operating at 80 kV.

Results Fluorescence measurement of inhibition—To test whether D-Trp-Aib can inhibit amyloid formation by molecules other than IAPP, Alzheimer's β-amyloid 1-40 (Aβ) served as a model system. As shown in FIG. 50, in the absence of inhibitor, Aβ showed a delayed phase in fibrillation of about 100 hours, followed by a rapid increase in fluorescence levels. In the presence of 10 μM EG30, the lag time was significantly increased and the overall fluorescence when plateau was reached was significantly lower than that observed without inhibitor.

  This further suggests that aromatic inhibitors may act as a common mechanism for common amyloid inhibitors and associations [Gazit (2002), FASEB J. et al. 16, 77-83].

  Ultrastructural analysis—To obtain information on the mechanism of inhibition, samples taken from the fluorescence assay during its termination assay were examined by electron microscopy (FIGS. 51a-c). Clear and well-defined amyloid fibrils were present in the Aβ sample without inhibitor (FIG. 51a). In contrast, in the presence of EG30, Aβ was detected as almost amorphous aggregates (FIG. 51b) or as split fibrils (FIG. 51c). This suggests that even those fibrils formed in the presence of inhibitors were short and probably dysfunctional.

  Thus, D-Trp-Aib compounds provide a unique combination of pharmaceutical properties in a very small molecule:

  1. Heteroaromatic interaction: The interaction between the aromatic recognition interface of the growing amyloid fibril (Gazit, 2002) and tryptophan indole is a direct and accurate block that blocks further homomolecular self-association of the growing chain Allows for oriented bonding.

  2. Aib conformational restriction: β-aminoisobutyric acid (Aib), an amino acid binding with exceptional geometric constraints, induces a very strong β-sheet breaking effect and stops amyloid fibril growth It is a key means. This β-breaking strategy shows clear advantages compared to the prior art (proline introduction) in terms of molecular size and complexity.

  3. Stable D-isomer conformation: Inhibitors are made from D-amino acids and achiral Aib moieties. This results in the formation of peptide bonds that cannot be cleaved, thus possibly resulting in high physiological stability.

  4). Peptide bond stacking: Regardless of its small size, typical peptide bonds (although they are isomerically stable) are retained in the molecule. The unique planar nature of such peptide bonds is due to their partially planar resonant structure with an exact geometric configuration consistent with β-chain interactions, due to the specific geometry of the molecules on the growing amyloid chain Allows limited stacking.

  5). Electrostatic repulsion: The presence of a charged end results in an electrostatic repulsion of additional bound monomers. Such repulsion is achieved by introducing charged aspartic acid in other peptide inhibitors [Soto et al. (2003), J. Biol. Biol. Chem 278: 13905]. This is because it is necessary to block the ends of these peptides to reduce proteolysis. The non-natural stable isomeric form of D-Trp-Aib allows the retention of charged ends within a minimal framework that yields significantly smaller molecules.

  6). Bulky hydrophobic moieties: Tryptophan indole groups provide unique bulky and hydrophobic properties in very small molecular systems. It is well established that the tryptophan moiety has the highest membrane partition coefficient. This property would have key advantages for oral bioavailability and blood brain barrier (BBB) transition.

  7). Antioxidant activity: Indole groups are also known to act as antioxidants by the removal of free radicals. Indeed, some drug candidates for treating AD are based on this property (eg, MindSet's indole-3-propionic acid). However, although such molecules are effective in protecting neurons from AD-related oxidative stress, they lack the unique fibrillation inhibitory properties of the D-Trp-Aib molecular frame.

  8). Small size: D-Trp-Aib is a significantly smaller active molecule. All of the unique properties described in the previous paragraphs (1-7) are maintained in molecules below 300 Da. The small size of this non-cleavable molecule suggests oral bioavailability, long half-life, and BBB migration while maintaining low immunogenicity.

  It will be appreciated that certain features of the invention described in separate embodiments for clarity may be provided in combination in a single embodiment. Conversely, various features of the invention described in a single embodiment for simplicity may be provided separately or in any suitable combination.

  Although the invention has been described with respect to specific embodiments thereof, it will be apparent that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, the spirit of the appended claims and their alternatives, modifications and variations that fall within the broad scope thereof are intended to be embraced therein. All publications, patents, and patent applications mentioned in this specification are to the full extent that each individual publication, patent, or patent application is specifically and individually incorporated herein by reference. This specification is incorporated by reference. Furthermore, the citation and description of any reference made in this application shall not be construed as an admission that the reference is available as prior art to the present invention.

It is a schematic diagram showing the self-association ability and its hydrophobicity of a group of peptides derived from a large number of amyloid proteins, as estimated using the Kyte and Dolittle scales. FIG. 2 schematically shows amyloid binding with inhibitory aromatic reagents Ro47-1816 / 001 (FIG. 2a), thioflavin T (FIG. 2b) and CR dye (FIG. 2c). 1 is a schematic diagram of primary sequence comparison between human IAPP and rodent IAPP and a synthetic peptide of the present invention. FIG. FIG. 5 is a graph showing the absorbance of light at 405 nm as a function of time during fibril formation, and thus the absorbance reflecting the aggregation kinetics of IAPP-derived peptides. 2 is a histogram showing the average particle size of associated IAPP peptides and derivatives as measured by light scattering. FIG. 5 is a photomicrograph showing the binding of Congo red to pre-associated IAPP peptide. 2 is an electron micrograph of “laying” IAPP peptides and derivatives. Nucleic acid sequence alignment of wild type hIAPP and corresponding sequences modified according to bacterial codon usage. FIG. 2 is a schematic diagram of the pMALc2x-NN vector used for cytoplasmic expression of the 48 kDa MBP-IAPP protein. Protein gel GelCode Blue staining showing bacterial expression and purification of MBP and MBP-IAPP fusion proteins. FIG. 10 is a dot blot image (FIG. 10a) showing the putative amyloidogenic sequence in hIAPP and its densitometric quantification (FIG. 10b). FIG. 4 is a graph showing the absorbance of light at 405 nm as a function of time during fibril formation, and thus the absorbance reflecting the aggregation kinetics of IAPP-derived peptides (SEQ ID NOs: 14-19). FIG. 5 is a photomicrograph showing the binding of Congo red to pre-associated IAPP peptide. 2 is an electron micrograph of a “laid” IAPP peptide. It is a graph which shows the secondary structure in an insoluble IAPP aggregate as measured by a Fourier transform infrared spectroscopy. The chemical structure of a previously reported amyloidogenic peptide fragment of medins [Haggqvist (1999), Proc. Natl. Acad. Sci. USA, 96: 8669-8684]. FIG. 6 is a graph showing the absorbance of light at 405 nm as a function of time during fibril formation, and thus the absorbance reflecting the aggregation kinetics of medin-derived peptides. 2 is an electron micrograph of a “laying” medin-derived peptide. It is a microscope picture which shows the coupling | bonding of the Congo red to the medin origin peptide which was made to associate previously. Figure 3 shows the effect of alanine mutations on the amyloidogenic properties of the amyloidogenic fragment of the hexapeptide of medin. Amino acid sequence of human calcitonin (FIG. 20a) and chemical structure of an amyloidogenic peptide fragment of human calcitonin (FIG. 20b). 2 is an electron micrograph of a “laying” calcitonin-derived peptide. It is a microscope picture which shows the coupling | bonding of Congo red to the calcitonin origin peptide made to associate beforehand. It is a graph which shows the secondary structure in an insoluble calcitonin aggregate as measured by a Fourier transform infrared spectroscopy. Figure 2 shows the effect of alanine mutations on amyloidogenic properties of amyloidogenic fragments of calcitonin pentapeptide. 2 is an electron micrograph showing self-association of “laying” lactotransferrin-derived peptide. 2 is an electron micrograph showing self-association of a “laying” serum amyloid A protein-derived peptide. 2 is an electron micrograph showing self-association of a “laying” BriL-derived peptide. 2 is an electron micrograph showing self-association of a “laying” gelsolin-derived peptide. 2 is an electron micrograph showing self-association of a “laying” serum amyloid P-derived peptide. 2 is an electron micrograph showing self-association of a “laying” immunoglobulin light chain-derived peptide. 2 is an electron micrograph showing self-association of a “laying” cystatin C-derived peptide. 2 is an electron micrograph showing self-association of a “laying” transthyretin-derived peptide. FIG. 2 is an electron micrograph showing self-association of a “laying” lysozyme-derived peptide. FIG. 2 is an electron micrograph showing self-association of a “laying” fibrinogen-derived peptide. 2 is an electron micrograph showing self-association of a “laying” insulin-derived peptide. 2 is an electron micrograph showing self-association of a “laying” prolactin-derived peptide. 2 is an electron micrograph showing self-association of a “laying” β2 microglobulin derived peptide. It is a graph which shows the effect | action of the inhibitory peptide with respect to the self-association of IAPP. FIG. 5 is a graph showing the absorbance of light at 405 nm as a function of time during fibril formation, and thus the absorbance reflecting the aggregation kinetics of the IAPP-derived peptide (SEQ ID NOs: 46-49). FIG. 2 shows a histogram showing the turbidity of an IAPP analog after aging for 7 days. It is the electron micrograph of the "laying down" IAPP analog. FIG. 5 shows binding of IAPP-NFGAILSS to an analog of the minimal amyloidogenic sequence SNNXGAILSS (X = any natural amino acid except cysteine). FIG. 43a shows possible configurations for all residues (yellow is fully possible, orange is partially possible), for L-proline (blue), and for the achiral Aib residue (magenta). Is a Ramachandran plot showing Figures 43b-c show the chemical structure of the long wild-type IAPP peptide (ANFLVH, SEQ ID NO: 124, Figure 43b) and its Aib modified structure peptide (Aib-NF-Aib-VH, SEQ ID NO: 125, Figure 43c). It is a schematic diagram shown. It is the electron micrograph of the "laying down" IAPP analog. FIG. 6 is a photomicrograph showing Congo red binding to pre-associated wild type and Aib modified IAPP peptides. FIG. 2 is a graph showing the secondary structure of insoluble wild type and Aib modified hIAPP aggregates as determined by Fourier Transform Infrared Spectroscopy (FI-IR). It is a graph which shows the inhibitory effect of the Aib modification peptide with respect to amyloid fibril formation. Figure 2 is a histogram showing the inhibitory effect of a short aromatic sequence (SEQ ID NOs: 112-123) on IAPP-self-association. FIG. 49a shows the results of the first round of selection of IAPP fibrillation inhibitor. FIG. 49b shows the results of the second round of selection of IAPP fibrillation inhibitor. FIG. 49c shows the results of the third round of selection of IAPP fibrillation inhibitor. 4 shows the results of the fourth round of selection of IAPP fibrillation inhibitors. It is a graph which shows inhibition of A (beta) (1-40) fibril formation by D-Trp-Aib. It is a microscope picture showing the inhibitory effect of D-Trp-Aib with respect to fibrillization of A (beta) so that it may be visualized by TEM.

SEQ ID NOs: 1-6, 8-49, 61-89, and 91-150 are synthetic peptide sequences.
SEQ ID NO: 7 is a consensus sequence.
SEQ ID NOs: 50-57 and 59-60 are the sequences of single-stranded DNA oligonucleotides.
SEQ ID NO: 58 is the sequence of a modified IAPP cDNA for expression in bacteria.
SEQ ID NO: 90 is a peptide array consensus sequence.

Claims (3)

A pharmaceutical composition for treating or preventing an amyloid-related disease comprising a peptide selected from the group consisting of SEQ ID NOs: 143 to 148 as an active ingredient and comprising a pharmaceutically acceptable carrier or diluent .   The pharmaceutical composition according to claim 1, wherein the peptide is a linear or cyclic peptide.   The pharmaceutical composition of claim 1, wherein the peptide is as set forth in SEQ ID NO: 145.